EP2734518B1 - Complexing agents and corresponding lanthanide complexes - Google Patents

Description

The present invention relates to complexing agents or ligands, lanthanide complexes, their use for labeling molecules and detecting them by time-resolved fluorescence techniques.

State of the art:

The lanthanide complexes have seen their use increase significantly in the last twenty years in the field of life sciences. These fluorescent compounds indeed have interesting spectroscopic characteristics, which make them markers of choice for detecting biological molecules. These fluorescent compounds are particularly suitable for use in conjunction with compatible fluorophores to carry out FRET measurements (the acronym for the term "Förster resonance energy transfer"), the application of which to study the interactions between biomolecules is exploited by several companies, including Cisbio Bioassays and its HTRF® product line. The relatively long lifetime of the lanthanide complexes also makes it possible to perform fluorescence measurements in time resolved, that is to say with a delay after excitation of the fluorophores, which makes it possible to limit the fluorescence interference due to the medium. measurement. This last characteristic is all the more useful as the measuring medium approaches a biological medium, which comprises many proteins whose fluorescence could interfere with that of the compounds studied.

Many lanthanide complexes have been described. Latva et al, for example, have disclosed 41 complexes of Eu (III) and Tb (III), whose luminescence they have studied ( Journal of Luminescence Volume 75, No. 2, September 1997, Pages 149-169 ). Compound 39 in particular consists of a 1,4,7-triazacyclononane ring (hereinafter "TACN"), the nitrogen atoms of which are substituted by chromophores derived from phenylethynylpyridine. Although the quantum yield of the complex consisting of this chromophore and Eu (III) is considered good by the authors, this complex is not suitable for coupling with a biomolecule. Moreover, the use of this compound in an aqueous medium can be problematic since it is very hydrophobic. Finally, the absorption of this complex is optimal at 315 nm, whereas the laser lamps often used in biological assays emit at the wavelength of 337 nm.

D'Aléo et al have described the synthesis of lanthanide complexes consisting of three ligands derived from dipicolonic acid ( Inorg Chem. 2008 Nov 17; 47 (22): 10258-68 ). One of these ligands (L1) consists of a dipicolinic acid molecule substituted by a phenylethynyl group, itself carrying a polyethylene glycol ether ether (hereinafter "PEG") on the phenyl group. According to the authors, the PEG group confers on this product a good solubility in aqueous media and in organic solvents. However, these complexes are not sufficiently stable in aqueous medium and are not usable in a bioconjugation reaction.

International patent application WO2005 / 058877 relates to lanthanide complexes, some of which are based on a TACN ring of which three nitrogen atoms are substituted by chromophores consisting of a pyridine derivative, in particular phenylpyridine. The inventors also propose to include in these compounds a reactive group to be able to easily conjugate with biomolecules. It is thus proposed to include this reactive group via a spacer arm either at a carbon of the TACN ring, or at the level of the pyridine of the chromophore.

Several other lanthanide complexes have been disclosed and some are commercially exploited: macropolycyclic cryptates of lanthanides ( EP 0 180 492 , EP 0 321 353 , EP 0 601 113 , WO 2001/96877 , WO2008 / 063721 ), the lanthanide complexes having a coumarin-derived unit linked to a diethylene triamine penta acid unit ( US 5,622,821 ), and those comprising pyridine derivatives ( US 4,920,195 , US 4,761,481 ), bipyridine ( US 5,216,134 ), or terpyridine ( US 4,859,777 , US 5,202,423 , US5,324,825 ).

The patent application WO89 / 01475 describes the preparation of tri-zoned macrocycles in which one of the carbon atoms carries a LZ group, and especially 2- (4-N-benzamidyl) butyl-1,4,7-triazacyclonane (intermediate 12). The synthesis of nitrogenous heteromacrocycles of which one of the carbon atoms is substituted is also described by Cox et al (J. Chem Soc., Perkin Trans, 1, 1990, 2567-2576 ) and Craig et al (J. Chem Soc., Chem., Commun., 1989, 794-796 ).

The present invention aims to overcome the disadvantages of the compounds of the prior art, and to provide fluorescent lanthanide complexes having a better gloss than the compounds of the prior art when they are excited at around 337 nm, if possible a good solubility in an aqueous medium, an emission spectrum adapted to their use in FRET experiments, as well as good practicality for the labeling of biomolecules.

Description of the invention

The aforementioned problems have been solved by means of complexing agents consisting of a tri-classified macrocycle (1,4,7-triazacyclononane, hereinafter 147 TACN, 1,5,9-triazacyclododecane, hereinafter 159 TACD, 1,4,8 -triazacyclodecane, hereinafter 148TACD or 1,4,8-triazacycloundecane, hereinafter 148TACU) whose nitrogen atoms are substituted by phenylethynylpyridine-type chromophores, these chromophores comprising from one to three groups affecting the electron density of the molecule (hereinafter group "O-donor", "S-donor", "NHCO-donor", "SCO-donor", "NHCS-donor", "SCS-donor" or lower alkyl) directly linked to the phenyl group, and optionally at least one polyethylene glycol (PEG) group which will give the molecule a good solubility in an aqueous medium. These compounds may also comprise a reactive group allowing their conjugation with a molecule to be labeled. The complexing agents according to the invention form stable complexes with lanthanides, and can be used to produce fluorescent conjugates of molecules of interest. The lanthanide complexes according to the invention have excellent photophysical properties, in particular as regards their quantum efficiency, the lifetime of their luminescence and their excitation spectrum, which is very well adapted to a laser excitation at approximately 337 nm. nm. The presence of three chromophores significantly increases the molar absorption coefficient (epsilon) and consequently the brightness of the complex. The brightness (quantum yield x molar absorption coefficient) of these complexes in biological media is also better than that of the compounds of the prior art.

COMPLEXING AGENTS

dans laquelle :

• soit a=b=c=1 (1,5,9-triazacyclododécane), soit a=b=c=0 (1,4,7-triazacyclononane), soit a=1 et b=c=0 (1,4,8-triazacyclodécane), soit a=b=1 et c=0 (1,4,8-triazacycloundécane) ;
• R₃, R₄ et R₅ sont chacun choisis parmi les atomes ou groupes suivants : H, -L-G ;
• Chrom₁, Chrom₂ et Chrom₃ sont des chromophores identiques ou différents et répondent tous trois à une seule des formules suivantes :
  
  
  
  dans lesquelles
  • - d est un nombre entier allant de 1 à 3
  • - chaque R₁ est choisi parmi les groupes suivants : -COOH, -PO(OH)R₆, R₆ représentant un groupe choisi parmi : phényle, benzyle, méthyle, éthyle, propyle, n-butyle, sec-butyle, isobutyle, tert-butyle;
  • - R₂₁ et R₂₂ représentent indépendamment l'un de l'autre un atome d'hydrogène ou un un alkyle linéaire ou ramifié comprenant de 1 à 6 atomes de carbone ;
  • - chaque R₂ est choisi parmi les groupes suivants :
    • groupes alkyles linéaires ou ramifiés comprenant de 1 à 6 atomes carbones éventuellement substitués par un groupe PEG ;
    • groupes O-donneurs choisis parmi: OH ; -OPhényle ; -O-CH₂-CO-N-Alk ; -O-CH₂-CO-O-Alk ; -O-CH₂-CO-NH ; -O-CH₂-CO-OH; O-CH₂-CO-N-LG ; -O-CH₂-CO-O-L-G -O-L-G ; -O-Alk; -O-PEG; le groupe de formule :
      
      dans laquelle z est un nombre entier allant de 1 à 5 ;
    • groupes S-donneurs choisis parmi : -S-L-G ; -S-Alk; -S-PEG; le groupe de formule :
      
      dans laquelle z est un nombre entier compris entre 1 et 5 ;
    • groupes NHCO-donneurs choisis parmi : -NHCO-L-G ; -NHCO(OAlk); -NHCO(NHAlk); -NHCO(NAIk1Alk2); -NHCO(SAlk); -NHCO(Alk); -NHCO-PEG; -NHCO-phényle ; un groupe de formule :
      
    • groupes SCO-donneurs choisis parmi : -SCO-L-G; -SCO(OAlk); -SCO(NHAlk); -SCO(NAlk₁Alk₂); -SCO(SAlk); -SCO(Alk) ; -SCO-PEG;
    • groupes NHCS-donneurs choisis parmi : -NHCS(OAlk); -NHCS(NHAlk); -NHCS(NAlk₁Aik₂); -NHCS(SAlk); -NHCS(Alk); -NHCS-PEG; -NHCS-L-G;
    • groupes SCS-donneurs choisis parmi : -SCS(OAlk); -SCS(NHAlk); -SCS(NAlk₁Alk₂); -SCS(SAlk); -SCS(Alk) ; -SCS-PEG; -SCS-L-G;
      • PEG est un groupe de formule-CH₂-(CH₂OCH₂)y-CH₂OCH₃, y étant un nombre entier allant de 1 à 5;
      • Alk, Alk1 et Alk2 sont chacun un alkyle linéaire ou ramifié en C₁-C₁₀ éventuellement substitué par un groupe -O-PEG ;
      • L est un bras d'espacement;
      • G est un groupe réactif permettant la liaison du complexe ou de l'agent complexant à une molécule à marquer;
    étant entendu que :
• lorsque R₂₂ et R₂₁ sont des groupes alkyles, d=1 et lorsque l'un des groupes R₂₂ ou R₂₁ est un groupe alkyle et que l'autre est un atome d'hydrogène, d=1 ou d= 2 ;
• lorsque R₂ comporte un groupe -L-G, R₃= R₄ = R₅ = H ;
• lorsque R₂ ne comporte pas de groupe -L-G, soit l'un des groupes R₃, R₄ et R₅ est un groupe
• L-G soit R₃ = R₄ = R₅ = H; et
• lorsque plusieurs groupes R₂ sont présents, au moins un est en position 4 du cycle benzénique.

The complexing agents according to the invention are the compounds of formula (I):

in which :

• either a = b = c = 1 (1,5,9-triazacyclododecane), or a = b = c = O (1,4,7-triazacyclononane), or a = 1 and b = c = 0 (1,4 8-triazacyclodecane), a = b = 1 and c = O (1,4,8-triazacycloundecane);
• R ₃ , R ₄ and R ₅ are each selected from the following atoms or groups: H, -LG;
• Chrom ₁ , Chrom ₂ and Chrom ₃ are identical or different chromophores and all three of them correspond to only one of the following formulas:
  
  
  
  in which
  • - d is an integer ranging from 1 to 3
  • each R ₁ is chosen from the following groups: -COOH, -PO (OH) R ₆ , R ₆ representing a group chosen from: phenyl, benzyl, methyl, ethyl, propyl, n- butyl, sec-butyl, isobutyl, tert- butyl;
  • - R ₂₁ and R ₂₂ represent independently of one another a hydrogen atom or a linear or branched alkyl comprising 1 to 6 carbon atoms;
  • each R ₂ is chosen from the following groups:
    • linear or branched alkyl groups comprising 1 to 6 carbon atoms optionally substituted with a PEG group;
    • O-donor groups selected from: OH; -Phenyl; -O-CH ₂ -CO-N-Alk; -O-CH ₂ -CO-O-Alk; -O-CH ₂ -CO-NH; -O-CH ₂ -CO-OH; O-CH ₂ -CO-N-LG; -O-CH ₂ -CO-OLG-OLG; -O-Alk; -O-PEG; the group of formula:
      
      wherein z is an integer from 1 to 5;
    • S-donor groups selected from: -SLG; -S-Alk; -S-PEG; the group of formula:
      
      wherein z is an integer from 1 to 5;
    • NHCO-donor groups selected from: -NHCO-LG; -NHCO (OAlk); -NHCO (NHAlk); -NHCO (NAIk1Alk2); -NHCO (Salk); -NHCO (Alk); -NHCO-PEG; -NHCO-phenyl; a group of formula:
      
    • SCO-donor groups selected from: -SCO-LG; -SCO (OAlk); -SCO (NHAlk); -SCO (NAlk ₁ Alk ₂ ); -SCO (Salk); -SCO (Alk); -SCO-PEG;
    • NHCS-donor groups selected from: -NHCS (OAlk); -NHCS (NHAlk); -NHCS (NAlk ₁ Aik ₂ ); -NHCS (Salk); -NHCS (Alk); -NHCS-PEG; -NHCS-LG;
    • SCS-donor groups selected from: -SCS (OAlk); -SCS (NHAlk); -SCS (NAlk ₁ Alk ₂ ); -SCS (Salk); -SCS (Alk); -SCS-PEG; -SCS-LG;
      • PEG is a group of formula -CH ₂ - (CH ₂ OCH ₂ ) y -CH ₂ OCH ₃ , where y is an integer from 1 to 5;
      • Alk, Alk1 and Alk2 are each linear or branched C ₁ -C ₁₀ alkyl optionally substituted with a -O-PEG group;
      • L is a spacer arm;
      • G is a reactive group for the binding of the complex or complexing agent to a molecule to be labeled;
    Being heard that :
• when R ₂₂ and R ₂₁ are alkyl groups, d = 1 and when one of R ₂₂ or R ₂₁ is alkyl and the other is hydrogen, d = 1 or d = 2;
• when R ₂ has a group -LG, R ₃ = R ₄ = R ₅ = H;
• when R ₂ does not have a group -LG, one of the groups R ₃ , R ₄ and R ₅ is a group
• LG is R ₃ = R ₄ = R ₅ = H; and
• when more than one R _{2 group} is present, at least one is in the 4-position of the benzene ring.

Preferably, Chrom ₁ , Chrom ₂ and Chrom ₃ are identical or different chromophores and all three of them correspond to only one of the following formulas:

dans laquelle :

• soit a=b=c=1 (1,5,9-triazacyclododécane), soit a=b=c=0 (1,4,7-triazacyclononane), soit a=1et b=c=0 (1,4,8-triazacyclodécane), soit a=b=1 et c=0 (1,4,8-triazacycloundécane) ;
• R₃, R₄ et R₅ sont chacun choisis parmi les atomes ou groupes suivants : H, -L-G ;
• Chrom₁, Chrom₂ et Chrom₃ sont des chromophores identiques ou différents de formule (II) :
  
  dans laquelle
  • d est un nombre entier allant de 1 à 3 ;
  • R₁ est choisi parmi les groupes suivants : -COOH, -PO(OH)R₆, R₆ représentant un groupe choisi parmi : phényle, benzyle, méthyle, éthyle, propyle, n-butyle, sec-butyle, isobutyle, tert-butyle, les groupes phényle et méthyle étant préférés;
  • chaque R₂ est choisi parmi les groupes suivants :
    • groupes alkyles linéaires ou ramifiés comprenant de 1 à 6 atomes de carbone éventuellement substitués par un groupe PEG ;
    • groupes O-donneurs choisis parmi : -O-L-G ; -O-Alk; -O-PEG; le groupe de formule :
      
      dans laquelle z est un nombre entier allant de 1 à 5 ;
    • groupes S-donneurs choisis parmi : -S-L-G ; -S-Alk; -S-PEG; le groupe de formule :
      
      dans laquelle z est un nombre entier allant de 1 à 5 ;
    • groupes NHCO-donneurs choisis parmi : -NHCO-L-G ; -NHCO(OAlk); -NHCO(NHAlk); -NHCO(NAlk₁Alk₂); -NHCO(SAlk); -NHCO(Alk); -NHCO-PEG; le groupe de formule :
      
    • groupes SCO-donneurs choisis parmi : -SCO-L-G; -SCO(OAlk); -SCO(NHAlk); -SCO(NAlk₁Alk₂); -SCO(SAlk); -SCO(Alk) ; -SCO-PEG;
    • groupes NHCS-donneurs choisis parmi : -NHCS(OAlk); -NHCS(NHAlk); -NHCS(NAlk₁Alk₂); -NHCS(SAlk); -NHCS(Alk); -NHCS-PEG; -NHCS-L-G ;
    • groupes SCS-donneurs choisis parmi : -SCS(OAlk); -SCS(NHAlk); -SCS(NAlk₁Alk₂); -SCS(SAlk); -SCS(Alk) ; -SCS-PEG; -SCS-L-G;
      • PEG est un groupe de formule -CH₂-(CH₂OCH₂)y-CH₂OCH₃, y étant un nombre entier allant de 1 à 5;
      • Alk, Alk1 et Alk2 sont chacun un alkyle linéaire ou ramifié en C₁-C₁₀ éventuellement substitué par un groupe -O-PEG ;
  • L est un bras d'espacement;
  • G est un groupe réactif permettant la liaison du complexe ou de l'agent complexant à une molécule à marquer;
  étant entendu que :
  • lorsque R₂ comporte un groupe -L-G, R₃ = R₄ = R₅ = H ;
  • lorsque R₂ ne comporte pas de groupe -L-G, soit l'un des groupes R₃, R₄ et R₅ est un groupe -L-G, soit R₃ = R₄ = R₅ = H; et
  • lorsque plusieurs groupes R₂ sont présents, au moins un est en position 4 du cycle benzénique.

The invention relates in particular to the complexing agents of formula (I):

in which :

• either a = b = c = 1 (1,5,9-triazacyclododecane), or a = b = c = O (1,4,7-triazacyclononane), or a = 1 and b = c = 0 (1,4, 8-triazacyclodecane), a = b = 1 and c = O (1,4,8-triazacycloundecane);
• R ₃ , R ₄ and R ₅ are each selected from the following atoms or groups: H, -LG;
• Chrom ₁ , Chrom ₂ and Chrom ₃ are identical or different chromophores of formula (II):
  
  in which
  • d is an integer from 1 to 3;
  • R ₁ is chosen from the following groups: -COOH, -PO (OH) R ₆ , R ₆ representing a group chosen from: phenyl, benzyl, methyl, ethyl, propyl, n- butyl, sec-butyl, isobutyl, tert - butyl, with phenyl and methyl being preferred;
  • each R ₂ is selected from the following groups:
    • linear or branched alkyl groups having 1 to 6 carbon atoms optionally substituted with a PEG group;
    • O-donor groups selected from: -OLG; -O-Alk; -O-PEG; the group of formula:
      
      wherein z is an integer from 1 to 5;
    • S-donor groups selected from: -SLG; -S-Alk; -S-PEG; the group of formula:
      
      wherein z is an integer from 1 to 5;
    • NHCO-donor groups selected from: -NHCO-LG; -NHCO (OAlk); -NHCO (NHAlk); -NHCO (NAlk ₁ Alk ₂ ); -NHCO (Salk); -NHCO (Alk); -NHCO-PEG; the group of formula:
      
    • SCO-donor groups selected from: -SCO-LG; -SCO (OAlk); -SCO (NHAlk); -SCO (NAlk ₁ Alk ₂ ); -SCO (Salk); -SCO (Alk); -SCO-PEG;
    • NHCS-donor groups selected from: -NHCS (OAlk); -NHCS (NHAlk); -NHCS (NAlk ₁ Alk ₂ ); -NHCS (Salk); -NHCS (Alk); -NHCS-PEG; -NHCS-LG;
    • SCS-donor groups selected from: -SCS (OAlk); -SCS (NHAlk); -SCS (NAlk ₁ Alk ₂ ); -SCS (Salk); -SCS (Alk); -SCS-PEG; -SCS-LG;
      • PEG is a group of the formula -CH ₂ - (CH ₂ OCH ₂ ) y -CH ₂ OCH ₃ , where y is an integer from 1 to 5;
      • Alk, Alk1 and Alk2 are each linear or branched C ₁ -C ₁₀ alkyl optionally substituted with a -O-PEG group;
  • L is a spacer arm;
  • G is a reactive group for the binding of the complex or complexing agent to a molecule to be labeled;
  Being heard that :
  • when R ₂ has a group -LG, R ₃ = R ₄ = R ₅ = H;
  • when R ₂ does not contain a group -LG, one of the groups R ₃ , R ₄ and R ₅ is a group -LG, ie R ₃ = R ₄ = R ₅ = H; and
  • when more than one R _{2 group} is present, at least one is in the 4-position of the benzene ring.

• soit chrom₁, chrom₂ et chrom₃ sont identiques et sont chacun substitués par un à trois groupes R₂ ne comportant pas de groupe -L-G, et les groupes R₃-R₅ sont des atomes d'hydrogène ;
• soit chrom₁, chrom₂ et chrom₃ sont identiques et sont substitués par un à trois groupes R₂ ne comportant pas de groupe -L-G, et un des groupes R₃-R₅ est un groupe L-G, les autres étant des atomes d'hydrogène ;
• soit deux des groupes chrom₁, chrom₂ et chrom₃ sont identiques et sont chacun substitués par un à trois groupes R₂ ne comportant pas de groupe -L-G et le troisième chromophore est substitué par un groupe R₂ comportant un groupe -L-G, les groupes R₃-R₅ sont des atomes d'hydrogène.

A preferred family of compounds of formula (I) include compounds where:

• either chrom ₁ , chrom ₂ and chrom ₃ are identical and are each substituted with one to three groups R ₂ having no -LG group, and the groups R ₃ -R ₅ are hydrogen atoms;
• either chrom ₁ , chrom ₂ and chrom ₃ are identical and are substituted with one to three groups R ₂ not having a -LG group, and one of the groups R ₃ -R ₅ is a group LG, the others being atoms of hydrogen;
• or two of the chrom ₁ , chrom ₂ and chrom _{3 groups} are identical and are each substituted with one to three R ₂ groups having no -LG group and the third chromophore is substituted with a group R ₂ having a -LG group, the R ₃ -R _{5 groups} are hydrogen atoms.

• composés de formule (I) caractérisés en ce que l'un des groupes R₂ comporte un groupe -L-G ;
• composés de formule (I) caractérisés en ce que l'un des groupes R₃, R₄ ou R₅ comporte un groupe -L-G ;
• composés de formule (I) caractérisés en ce qu'il ne comporte aucun groupe -L-G.

The following sub-families of complexing agents are preferred:

• compounds of formula (I) characterized in that one of the groups R ₂ has a group -LG;
• compounds of formula (I) characterized in that one of the groups R ₃ , R ₄ or R ₅ has a group -LG;
• compounds of formula (I) characterized in that it contains no -LG group.

• Ceux qui sont caractérisés en ce que a=b=c=1 (1,5,9-triazacyclododécane, 159TACD) ;
• Ceux qui sont caractérisés en ce que a=b=c=0 (1,4,7-triazacyclononane, 147TACN) ;
• Ceux qui sont caractérisés en ce que a=1 et b=c=0 (1,4,8-triazacyclodécane, 148TACD) ;
• Ceux qui sont caractérisés en ce que a=b=1 et c=0 (1,4,8-triazacycloundécane, 148TACU).

Among the compounds of formula (I) and those belonging to the abovementioned sub-families, the following compounds are preferred:

• Those characterized in that a = b = c = 1 (1,5,9-triazacyclododecane, 159TACD);
• Those characterized in that a = b = c = O (1,4,7-triazacyclononane, 147TACN);
• Those characterized in that a = 1 and b = c = O (1,4,8-triazacyclodecane, 148TACD);
• Those characterized in that a = b = 1 and c = 0 (1,4,8-triazacycloundecane, 148TACU).

• Les composés caractérisés en ce que les groupes R₁ des chromophores chrom₁, chrom₂ et chrom₃ sont des groupes -COOH ;
• Les composés caractérisés en ce que les groupes R₁ des chromophores chrom₁, chrom₂ et chrom₃ sont des groupes -PO(OH)R₆ ;
• Les composés caractérisés en ce que le groupe R₁ du chromophore chrom₁ est un groupe -COOH et les groupes R₁ des chromophores chrom₂ et chrom₃ sont des groupes -PO(OH)R₆ ;
• Les composés caractérisés en ce que le groupe R₁ du chromophore chrom₁ est un groupe -PO(OH)R₆, et les groupes R₁ des chromophores chrom₂ et chrom₃ sont des groupes -COOH.

Among the compounds of formula (I) and those belonging to the abovementioned sub-families, the compounds having the following characteristics are also preferred:

• The compounds characterized in that the R ₁ groups of the chromophores chrom ₁ , chrom ₂ and chrom ₃ are -COOH groups;
• The compounds characterized in that the R ₁ groups of chromophores chrom ₁ , chrom ₂ and chrom ₃ are -PO (OH) R _{6 groups} ;
• The compounds characterized in that the group R ₁ of the chromophore chromium ₁ is a -COOH group and the groups R ₁ of the chromophores chrom ₂ and chrom ₃ are -PO (OH) R _{6 groups} ;
• The compounds characterized in that the group R ₁ of chromophore chromium ₁ is a group -PO (OH) R ₆ , and the groups R ₁ of chromophores chrom ₂ and chrom ₃ are -COOH groups.

• les composés caractérisés en ce que les groupes R₂ sont des groupes alkyles linéaires ou ramifiés comprenant de 1 à 6 atomes carbones éventuellement substitués par un groupe PEG ;
• les composés caractérisés en ce que les groupes R₂ sont des groupes O-donneurs choisis parmi : -O-L-G ; -O-Alk; -O-PEG; le groupe de formule :
  
• les composés caractérisés en ce que les groupes R₂ sont des groupes S-donneurs choisis parmi : -S-L-G ; -S-Alk; -S-PEG; le groupe de formule :
  
• les composés caractérisés en ce que les groupes R₂ sont des groupes NHCO-donneurs choisis parmi : -NHCO-L-G ; -NHCO(OAlk); -NHCO(NHAlk); -NHCO(NAlk₁Alk₂); -NHCO(SAlk); -NHCO(Alk); -NHCO-PEG; le groupe de formule :
  
• les composés caractérisés en ce que les groupes R₂ sont des groupes SCO-donneurs choisis parmi : -SCO-L-G; -SCO(OAlk); -SCO(NHAlk); -SCO(NAlk₁Alk₂); -SCO(SAlk); -SCO(Alk) ; -SCO-PEG;
• les composés caractérisés en ce que les groupes R₂ sont des groupes NHCS-donneurs choisis parmi -NHCS(OAlk); -NHCS(NHAlk); -NHCS(NAlk₁Alk₂); -NHCS(SAlk); -NHCS(Alk); -NHCS-PEG; -NHCS-L-G ;
• les composés caractérisés en ce que les groupes R₂ sont des groupes SCS-donneurs choisis parmi : -SCS(OAlk); -SCS(NHAlk); -SCS(NAlk₁Alk₂); -SCS(SAlk); -SCS(Alk) ; -SCS-PEG; -SCS-L-G.

One of the essential technical characteristics of the compounds according to the invention is the presence, on the chromophores, of R ₂ groups which affect the electron density of the molecule and contribute to the good spectroscopic properties of these compounds. Thus, among the compounds of formula (I) and those belonging to the abovementioned sub-families, the compounds whose donor groups R ₂ are the following are preferred:

• the compounds characterized in that the R ₂ groups are linear or branched alkyl groups comprising from 1 to 6 carbon atoms optionally substituted with a PEG group;
• the compounds characterized in that the R ₂ groups are O- donor groups selected from : -OLG; -O-Alk; -O-PEG; the group of formula:
  
• the compounds characterized in that the R ₂ groups are S-donor groups selected from: -SLG; -S-Alk; -S-PEG; the group of formula:
  
• the compounds characterized in that the R ₂ groups are NHCO-donor groups selected from : -NHCO-LG; -NHCO (OAlk); -NHCO (NHAlk); -NHCO (Alk ₂ Nalk _1); -NHCO (Salk); -NHCO (Alk); -NHCO-PEG; the group of formula:
  
• the compounds characterized in that the R ₂ groups are SCO-donor groups selected from: -SCO-LG; -SCO (OAlk); -SCO (NHAlk); -SCO (NAlk ₁ Alk ₂ ); -SCO (Salk); -SCO (Alk); -SCO-PEG;
• the compounds characterized in that the R ₂ groups are NHCS-donor groups selected from -NHCS (OAlk); -NHCS (NHAlk); -NHCS (NAlk ₁ Alk ₂ ); -NHCS (Salk); -NHCS (Alk); -NHCS-PEG; -NHCS-LG;
• the compounds characterized in that the R ₂ groups are SCS-donor groups selected from: -SCS (OAlk); -SCS (NHAlk); -SCS (NAlk ₁ Alk ₂ ); -SCS (Salk); -SCS (Alk); -SCS-PEG; -SCS-LG.

• un seul groupe R₂ en position 4 du groupe phényle, ou bien
• un groupe R₂ en position 4 et un groupe R₂ en position 3 du groupe phényle, ou bien
• un groupe R₂ en position 4 et un groupe R₂ en position 5 du groupe phényle, ou bien
• un groupe R₂ en position 4 et un groupe R₂ en position 2 du groupe phényle, ou bien
• un groupe R₂ en position 4 et un groupe R₂ en position 6 du groupe phényle, ou bien
• un groupe R₂ en position 4, un groupe R₂ en position 3 et un groupe R₂ en position 5 du groupe phényle, ou bien
• un groupe R₂ en position 4, un groupe R₂ en position 2 et un groupe R₂ en position 6 du groupe phényle.

The compounds according to the invention comprise from one to three groups R ₂ per chromophore, and preferably comprise:

• a single R ₂ group at the 4-position of the phenyl group, or
• a group R ₂ in position 4 and a group R ₂ in position 3 of the phenyl group, or
• a group R ₂ in position 4 and a group R ₂ in position 5 of the phenyl group, or
• a group R ₂ in position 4 and a group R ₂ in position 2 of the phenyl group, or
• a group R ₂ in position 4 and a group R ₂ in position 6 of the phenyl group, or
• a group R ₂ in position 4, a group R ₂ in position 3 and a group R ₂ in position 5 of the phenyl group, or
• a group R ₂ at the 4-position, a group R ₂ at the 2-position and a group R ₂ at the 6-position of the phenyl group.

When several R ₂ groups are present on a chromophore, they are preferably identical.

Finally, the preceding compounds characterized in that they comprise at least one PEG group are also preferred.

Table 1 represents various families of preferred compounds according to the invention: these compounds correspond to the general formula (I) and some of their characteristics are indicated in the first column (tri-classified macrocycle type) and the first line (nature of the R substituents). ₂ and R ₁ chromophores). Each "+" sign represents a family of preferred compounds. Thus, the box corresponding to the intersection of the first column and the first line designates the family of compounds comprising a ring 147TACN, the chromophores of which carry R ₂ groups which are alkylating groups and whose three R ₁ groups are COOH groups. The abbreviation "R ₁ : (COOH) 2 (POOHR ₆ )" means that one of R ₁ is COOH and the other two R ₁ are POOHR ₆ ).

• composés caractérisés en ce que l'un des groupes R₂ comporte un groupe -L-G ;
• composés caractérisés en ce que l'un des groupes R₃, R₄ ou R₅ comporte un groupe - L-G;
• composés caractérisés en ce qu'ils ne comportent aucun groupe -L-G.

For each of the families of compounds in Table 1, the following subfamilies are preferred:

• compounds characterized in that one of the groups R ₂ has a group -LG;
• compounds characterized in that one of the groups R ₃ , R ₄ or R ₅ has a group - LG;
• compounds characterized in that they contain no -LG group.

• un seul groupe R₂ en position 4 du groupe phényle, ou bien
• un groupe R₂ en position 4 et un groupe R₂ en position 3 du groupe phényle, ou bien
• un groupe R₂ en position 4 et un groupe R₂ en position 5 du groupe phényle, ou bien
• un groupe R₂ en position 4 et un groupe R₂ en position 2 du groupe phényle, ou bien
• un groupe R₂ en position 4 et un groupe R₂ en position 6 du groupe phényle, ou bien
• un groupe R₂ en position 4, un groupe R₂ en position 3 et un groupe R₂ en position 5 du groupe phényle, ou bien
• un groupe R₂ en position 4, un groupe R₂ en position 2 et 1 groupe R₂ en position 6 du groupe phényle.

Moreover, for each of the families of Table 1, the compounds comprise from one to three groups R ₂ per chromophore, and preferably comprise:

• a single R ₂ group at the 4-position of the phenyl group, or
• a group R ₂ in position 4 and a group R ₂ in position 3 of the phenyl group, or
• a group R ₂ in position 4 and a group R ₂ in position 5 of the phenyl group, or
• a group R ₂ in position 4 and a group R ₂ in position 2 of the phenyl group, or
• a group R ₂ in position 4 and a group R ₂ in position 6 of the phenyl group, or
• a group R ₂ in position 4, a group R ₂ in position 3 and a group R ₂ in position 5 of the phenyl group, or
• a group R ₂ in position 4, a group R ₂ in position 2 and a group R ₂ in position 6 of the phenyl group.

The compounds of Table 1 characterized in that they comprise at least one PEG group are also preferred.

Finally, when the compounds of Table 1 have an R ₆ group, it is preferably a methyl or phenyl group.

The subject of the invention is also the compounds, ligands and complexes described in Table 2.

The reactive group G carried by a spacer arm L, makes it possible to couple the compounds according to the invention with a species that it is desired to make fluorescent, for example an organic molecule, a peptide or a protein. The conjugation techniques of two organic molecules are based on the use of reactive groups and fall within the general knowledge of those skilled in the art. These conventional techniques are described for example in Bioconjugate Techniques, Hermanson WG, Academic Press, Second Edition 2008, p. 169-211 .

dans lesquels w varie de 0 à 8 et v est égal à 0 ou 1, et Ar est un hétérocycle à 5 ou 6 chaînons saturé ou insaturé, comprenant 1 à 3 hétéroatomes, éventuellement substitué par un atome d'halogène.Typically, the reactive group is an electrophilic or nucleophilic group that can form a covalent bond when brought into contact with a suitable nucleophilic or electrophilic group, respectively. The conjugation reaction between a compound according to the invention comprising a reactive group and an organic molecule, a peptide or a protein bearing a functional group results in the formation of a covalent bond having one or more atoms of the reactive group.
Preferably, the reactive group G is a group derived from one of the following compounds: an acrylamide, an activated amine (for example a cadaverine or an ethylenediamine), an activated ester, an aldehyde, an alkyl halide, a anhydride, aniline, azide, aziridine, carboxylic acid, diazoalkane, haloacetamide, halotriazine, such as monochlorotriazine, dichlorotriazine, hydrazine (including hydrazides), imido ester, isocyanate, isothiocyanate , a maleimide, a sulfonyl halide, or a thiol, a ketone, an amine, an acid halide, a hydroxysuccinimidyl ester, a hydroxysulfosuccinimidyl ester, an azidonitrophenyl, an azidophenyl, a 3- (2-pyridyl) dithio) -propionamide, a glyoxal, a triazine, an acetylenic group, and in particular the groups of formula:

wherein w is from 0 to 8 and v is 0 or 1, and Ar is a saturated or unsaturated 5- or 6-membered heterocycle comprising 1 to 3 heteroatoms, optionally substituted by a halogen atom.

Preferably, the reactive group G is a carboxylic acid, an amine, a succinimidyl ester, a haloacetamide, a hydrazine, an isothiocyanate, a maleimide group or an aliphatic amine.

These reactive groups can be directly linked to the complexing agent by a covalent bond or via a spacer arm advantageously constituted by a radical organic divalent, selected from linear or branched C ₁ -C ₂₀ alkylene groups, optionally containing one or more double or triple bonds; C ₅ -C ₈ cycloalkylene groups and C ₆ -C ₁₄ arylene groups, said alkylenes, cycloalkylenes or arylenes optionally containing one or more heteroatoms, such as oxygen, nitrogen, sulfur, phosphorus or one or more carbamoyl or carboxamido groups, and said alkylene, cycloalkylene or arylene groups being optionally substituted with C ₁ -C ₈ alkyl, C ₆ -C ₁₄ aryl, sulphonate or oxo groups.

1. 1)
   
2. 2)
   
3. 3)
   
4. 4)
   
5. 5)
   
6. 6)
   
7. 7)
   
8. 8)
   
9. 9)
   
10. 10)
    
11. 11)
    
12. 12)
    
13. 13)
    

dans lesquels n, m, p, r sont des nombres entiers de 1 à 16, de préférence de 1 à 5.In particular, the spacer arms may be chosen from the following divalent groups:

1. 1)
   
2. 2)
   
3. 3)
   
4. 4)
   
5. 5)
   
6. 6)
   
7. 7)
   
8. 8)
   
9. 9)
   
10. 10)
    
11. 11)
    
12. 12)
    
13. 13)
    

in which n, m, p, r are integers from 1 to 16, preferably from 1 to 5.

Preferably, the group -LG consists of a reactive group G chosen from: a carboxylic acid, an amine, a succinimidyl ester, a haloacetamide, a hydrazine, an isothiocyanate, a maleimide group, an aliphatic amine, and a spacer arm L consisting of an alkylene chain comprising 1 to 5 carbon atoms. Even more preferably, the group -LG is an amine carried by an alkylene chain comprising from 1 to 5 carbon atoms.

An organic molecule, a peptide or a protein that can be labeled with a compound according to the invention will therefore comprise a functional group with which the reactive group of the lanthanide complex or of the complexing agent will react. For example, the organic molecule, protein or peptide has one of the following groups: amine, amide, thiol, alcohol, aldehyde, ketone, hydrazine, hydroxylamine, secondary amine, halide, epoxide, ester (alkyl carboxylate), acid carboxylic groups, groups having double bonds or a combination of these functional groups. Amino groups or thiols naturally present on proteins are often used to proceed with the labeling of these molecules.

COMPLEX

The invention also relates to lanthanide complexes consisting of a lanthanide atom complexed with a complexing agent as described above, the lanthanide being chosen from: Eu ³⁺ , Tb ³⁺ , Gd ³⁺ , Dy ³⁺ , Nd ³⁺ , Er ³⁺ . Preferably, the lanthanide is Tb ³⁺ or Eu ³⁺ and even more preferably Eu ³⁺ .

These complexes are prepared by contacting the complexing agents according to the invention and a lanthanide salt. Thus the reaction between an equivalent of complexing agent and 1 to 5 equivalents of lanthanide salt (europium or terbium in the form of chlorides, acetates or triflates) in a solvent (acetonitrile, methanol or other solvent compatible with these salts) at reflux for several hours leads to the corresponding complex.

As indicated previously, the fluorescent complexes obtained have excellent photophysical properties, in particular with regard to their quantum efficiency, the lifetime of their luminescence and their excitation spectrum, which is very well adapted to a laser excitation at approximately 337 nm. nm. Moreover, the distribution of the bands of their emission spectra is centered around 620 nm, thus conferring on the complexes exceptional and very favorable properties in the use of FRET with cyanine or allophycocyanin type acceptors (such as XL665 marketed by Cisbio Bioassays). ). Due to the high stability of these complexes in biological media containing divalent cations (Mn ²⁺ , Ca ²⁺ , Mg ²⁺ ...) or EDTA, their luminescence remains excellent compared to the complexes of the art. prior.

Conjugates

The complexing and complexing agents according to the invention comprising a reactive group are particularly suitable for labeling organic or biological molecules comprising a functional group capable of reacting with the reactive group to form a covalent bond. Thus the invention also relates to the use of complexes for labeling biological molecules (proteins, antibodies, enzymes, hormones, etc.).

The invention also relates to the molecules labeled with a complex according to the invention. All organic or biological molecules can be conjugated with a complex according to the invention if they have a functional group capable of reacting with the reactive group. In particular, the conjugates according to the invention comprise a complex according to the invention and a molecule chosen from: an amino acid, a peptide, a protein, an antibody, a sugar, a carbohydrate chain, a nucleoside, a nucleotide, an oligonucleotide an enzyme substrate (in particular a suicide enzyme substrate such as a benzylguanine or a benzylcytosine (substrates of the enzymes sold under the names Snaptag and Cliptag), a chloroalkane (substrate of the enzyme marketed under the name Halotag); coenzyme A (substrate of the enzyme known under the name ACPtag or MCPtag).

SYNTH ESE

The preparation of the complexing agents (ligands) and complexes according to the invention is described schematically below, and in more detail in the experimental part.

Synthesis of the macrocycles 1,4,7-triazacyclononane (147TACN), 1,5,9-triazacyclododecane (TACD), 1,4,8-triazacyclodecane (1,4,8TACD) and 1,4,8-triazacycloundecane (148TACU)

Several processes for synthesizing 147TACN and 159TACD cycles have been described and the trihydrochloride salts of 147TACN and 159TACD are commercially available. The azamacrocycles 148TACD and 148TACU can be prepared using the procedure described by Anders et al (European Journal Inorganic Chemistry (2006) 1444-1455 ).

When the 3 chromophores grafted on the macrocycle are not identical, that is to say when one of them carries a -LG reactive group to allow the conjugation of the product with a compound to be labeled, the synthesis of the products according to the invention requires the use of tri-zoned macrocycles of which 1 or 2 secondary amines are protected by protecting groups. Parker et al have described the synthesis of TACN compounds of which one of the amines is protected by a benzoyl group ( Journal Chemical Society Perkin trans 1 (1990) 2567 ), however the deprotection of the amine is not compatible with the synthesis of the compounds according to the invention.

The compounds 147TACN, 159TACD, 148TACD and 148TACU monoprotected according to the invention have an amine protected by a group resistant to hydrogenation, for example by a carbamate group (Boc). These compounds are not commercially available, the elements published in the literature do not allow their synthesis (absence of experimental procedures) and it is also impossible to prepare them directly and with a suitable yield both the formation of macrocycles di- and trisubstituted is favored ( Kovacs et al. Chemical Society Journal, Chemical Commmunication 36 (1995) 9269-9272 ). In view of these technical problems, another strategy has been developed (SCHEMA 4). It is based on the preference of tri-classified macrocycles (147TACN, 159TACD, 148TACD and 148TACU) for disubstitution by first protecting two amines of the macrocycles with a protective group P, orthogonal to the Boc group or to any other group resistant to hydrogenation, that is to say whose deprotection conditions are different from those of the Boc group. The choice of the group P was focused on the Cbz protecting group as proposed in the literature using Cbz-ON (2-benzyloxycarbonyloxyimino) -2-phenylacetonitrile as precursor). However, since this compound is not commercially available, the synthesis was prepared using one of its analogues, Moz-ON ((2- (4-methoxybenzyloxycarbonyloxyimino) -2-phenylacetonitrile) which is available to it.

As described with Cbz-ON, two equivalents of Moz-ON were condensed on the tri-nitrogen macrocycle to give the disubstituted compounds with suitable yields. The purification of these products proved to be delicate because the Moz group is sensitive to acidity. Even that of silica is sufficient to cause degradation of the products during purification on a chromatography column. To avoid this degradation, the media The reactants were purified on a column of neutral alumina, which led, for example, to compounds 28a and 28b with yields of 74% and 34%, respectively. In the next step, the Boc group is introduced using N- ( tert- butoxycarbonyloxy) succinimide to give, for example, the trisubstituted triazotized macrocycles 29a and 29b. Finally, the amines carrying the Moz groups are deprotected by hydrogenolysis. The use of 10% Pd / C as a catalyst did not make it possible to deprotect the amines when the reaction medium is hydrogenated for 48 hours at 3.45 bars of pressure. On the other hand, the use of the Pearlman catalyst (Pd (OH) ₂ / C) made it possible to obtain mono-protected triazotized macrocycles. To easily isolate these products, the hydrochloride salts were formed by adding a small amount of cold hydrochloric acid, the latter being collected by precipitation in diethyl ether. This methodology now makes it possible to prepare, on a scale of several grams, the compounds (147TACN, 159TACD, 148TACD and 148TACU) monoprotected with a Boc group, for example 30a and 30b.

1. (i) protection de deux des trois amines du macrocycle triazoté par un groupe protecteur Gp₁ sensible à l'hydrogénation ;
2. (ii) purification des composés disubstitués sur colonne choisie parmi : une colonne d'alumine basique ; une colonne de silice ayant subi un traitement préalable avec de la triéthylamine ou de l'ammoniac; une colonne d'alumine basique ou neutre, cette dernière étant préférée ;
3. (iii) protection de la troisième amine dudit macrocycle triazoté par un deuxième groupe protecteur Gp₂ insensible à l'hydrogénation ;
4. (iv) déprotection des amines protégées par les groupes protecteurs Gp₁ par hydrogénation, de préférence avec le catalyseur de Pearlman ;
5. (v) purification des produits monoprotégés, par exemple par formation de sels de dihydrochlorure par ajout d'acide chlorhydrique à froid et précipitation dans l'éther diéthylique.

The method for synthesizing triprotected macrocycles monoprotected according to the invention thus comprises the following steps:

1. (i) protecting two of the three amines of the tri-nitrided macrocycle with a hydrogen-sensitive protecting group Gp ₁ ;
2. (ii) purification of the disubstituted compounds on a column chosen from: a basic alumina column; a silica column pretreated with triethylamine or ammonia; a column of basic or neutral alumina, the latter being preferred;
3. (iii) protecting the third amine of said tri-nitrogen macrocycle with a second protective group Gp ₂ insensitive to hydrogenation;
4. (iv) deprotecting the amines protected by the protecting groups Gp ₁ by hydrogenation, preferably with the Pearlman catalyst;
5. (v) purification of monoprotected products, for example by formation of dihydrochloride salts by addition of cold hydrochloric acid and precipitation in diethyl ether.

Gp ₁ and Gp ₂ are selected from amine protecting groups, which are known to those skilled in the art as described in Protecting Groups PJ Kocieński, Corrected Edition (2000) 185-244 Carbobenzyloxy (Cbz), p- Methoxybenzylcarbonyl (Moz) tert- Butyloxycarbonyl (Boc), 9-Fluorenylmethyloxycarbonyl (Fmoc), Acetyl (Ac), Benzoyl (Bz), Benzyl (Bn), p-Methoxybenzyl (PMB), 3, 4-Dimethoxybenzyl (DMPM), p-methoxyphenyl (PMP), Tosyl (Ts), Nosyl (Ns), trifluoroacetamides, alkoxycarbonyls, allyloxycarbonyl (Aloc), 2- (trimethylsilyl) ethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl (Troc) 2- (trimethylsilyl) ethoxycarbonyl (Teoc), B- (trimethylsilyl) ethanesulfonyl (SES), benzhydryl, trityl, 9-phenylfluorenyl and N-silyl imines.

Gp ₁ is preferably a Cbz or Moz group.

With regard to Gp ₂ , the groups Fmoc, Tosyl, Nosyl and Boc (trimethylsilyloxy) are preferred, the Boc group being even more preferred.

dans laquelle a=b=c=0 ou a=b=c=1. Le composé dans lequel Gp₂ est le groupe Boc est préféré. On comprend à la lecture de la méthode de synthèse décrite juste ci-dessus, et en particulier de l'étape (v), que les macrocycles triazotés monoprotégés selon l'invention se présentent sous la forme de sel de dihydrochlorure.The compound obtained is a compound of the following formula:

where a = b = c = 0 or a = b = c = 1. The compound in which Gp ₂ is the Boc group is preferred. It will be understood from reading the synthesis method described just above, and in particular from step (v), that the tri-protected macrocycles monoprotected according to the invention are in the form of dihydrochloride salt.

The preparation of the complexing agents of formula (I) in which R ₃ , R ₄ or R ₅ is a group -LG requires the use of particular macrocycles whose carbon 2 or 3 is substituted by the -LG group, or by a precursor of this group, especially a precursor in which the reactive group is in protected form. The synthesis of some of these azamacrocycles is described in particular in the international patent application WO 2005/058877 , as well as Hovinen et al (Tetrahedron Letters 46 (2005) 4387-4389 ). The methodology developed in these two references is based on the condensation between a trinosylated derivative and a diol using a Mitsunobu reaction. However, this method has the drawback of generating a large number of by-products that are not always easy to separate from the reaction medium, but above all does not allow the synthesis of functionalized 147TACN-type azamacrocycles as described in the Honiven article. Another strategy in softer conditions has been published by the Wu et al (Synthetic Communications, 34 (2004) 845-851 , Synthetic Communications 25 (1995) 1427-1437 , Chinese Journal of Chemistry, 16 (1998) 538-541 ) for obtaining the azamacrocycles by condensing a tritosyl derivative on the corresponding diol with yields close to 50%. A major disadvantage of this second methodology lies in the deprotection of the tosyle groups which is generally carried out under very harsh conditions (100 ° C., hydrobromic acid, in 33% acetic acid, or at 100 ° C. in the acid). concentrated sulfuric acid 96%).

The complexing agents whose group -LG is bound to the macrocycle have thus been prepared according to a strategy based on the condensation of a trinosylated derivative with a dibrominated derivative in which using cesium carbonate as a base in acetonitrile. The experimental conditions used are close to those described by Fukuyama et al (Tetrahedron, 58 (2002) 6267-6276 ) and are described in SCHEME 4 and the experimental part.

Synthesis of chromophores

The chromophores may be manufactured according to an approach as shown in Scheme A, and in which the groups R ₁ and R ₂ will optionally be protected, depending on their nature, especially if they are carboxylate groups, phosphates or reactive groups.

• Picot et al (Inorganic Chemistry 46 (2007), 2659-2665 : chromophore phényléthynylpyridine, groupe hexyloxy sur le groupement phényle ;
• Picot et al (Journal of American Chemical Society 130 (2008) 1532-1533 ): chromophore phényléthynylpyridine portant un groupe tris(triéthylèneglycol)phényl assurant la solubilité des composés ; et
• D'Aléo et al ( Sensitization of Eu(III) luminescence by donor-phenylethynyl-functionalized DTPA and DO3A macrocycles, C.R. Chimie 2010 ): phényléthynylpyridine portant un groupe OPeg).

This approach has been used for the synthesis of chromophores similar to those used for the preparation of the complexing agents according to the invention, for example by:

• Picot et al (Inorganic Chemistry 46 (2007), 2659-2665 : phenylethynylpyridine chromophore, hexyloxy group on the phenyl group;
• Picot et al (Journal of the American Chemical Society 130 (2008) 1532-1533 ): chromophore phenylethynylpyridine carrying a tris (triethylene glycol) phenyl group ensuring the solubility of the compounds; and
• Aléo et al (Sensitization of Eu (III) luminescence by donor-phenylethynyl-functionalized DTPA and DO3A macrocycles, CR Chemistry 2010 ): phenylethynylpyridine carrying a group OPeg).

The preparation of the reagents necessary for this synthesis is a general knowledge of those skilled in the art, when these reagents are not available commercially.
The synthesis of substituted alkynes is described in SCHEMES 1 and 13, as well as in the experimental part.
The synthesis of "carboxylate" chromophores is described in SCHEME 2 and in the experimental part.

The synthesis of chromophores "phosphinates" is described in SCHEME 3 and in the experimental part.

Grafting of chromophores on the macrocycle

The complexing agents of formula (I) having no -LG reactive group can be synthesized by alkylation of a tri-nitrogen macrocycle with a desired chromophore derivative (Scheme B) comprising a releasable group, such as a mesylate group ( -OMs), triflate (-OTf), tosylate (-OTs), or a chlorine, bromine or iodine atom. It may be necessary to protect the R ₁ groups that may react during alkylation, solubility problems or even polarity. For example, when R ₁ is a carboxylate or phosphinate group, corresponding ester derivatives may be used.

This synthesis is described in more detail in SCHEME 5 and the experimental part.

The same approach is used when the -LG group is directly linked to the macrocycle, for example for the complexing agents of formula (I) in which one of the groups R ₃ , R ₄ or R ₅ is a -LG group.

(R'₂ est tel que défini pour R₂ ci-dessus).The preparation of complexes in which one of the chromophores comprises a -LG reactive group can be carried out in the same way, by alkylation of a tri-amino macrocycle containing a protected amine function, prepared for example by a process such as that described above. Once the two chromophores grafted onto the macrocycle, the third chromophore bearing the -LG group (included in the R _{2 'group} ) may in turn be grafted after deprotection of the protected amine (scheme C).

(R ' ₂ is as defined for R ₂ above).

This synthesis is described in more detail in SCHEMES 6, 7, 8, 9 and the experimental part.

(R'₂ est tel que défini pour R₂ ci-dessus).An inverse strategy can be envisaged in which a chromophore comprising a masked LG reactive group is first introduced onto a diprotected azamacrocycle using an alkylation reaction. The two protective groups of the macrocycle orthogonal to the group protecting the LG reactive function are removed and the other two chromophores are then introduced by simple alkylation leading to the complexing agent skeleton (Scheme D).

(R ' ₂ is as defined for R ₂ above).

The synthesis of the compounds according to the invention is accurately detailed in the following experimental part.

Description of the Figures

• The Figure 1 represents the luminescence of the compounds of the invention in the presence of divalent cations or EDTA.
• The Figure 2 represents the excitation spectrum of the compounds of the invention in HEPES buffer.

Abbreviations used:

• Ms: Mesyl
• Boc: tert -Butyloxycarbonyl
• PEG: polyethylene glycol
• NHS: N-hydroxysuccinimide
• NMR: nuclear magnetic resonance
• TLC: Thin layer chromatography
• DMF: dimethylformamide
• THF: tetrahydrofuran
• DCM: dichloromethane
• HPLC: high performance liquid chromatography
• HRMS: high resolution mass spectroscopy
• TEA / And ₃ N: triethylamine
• DIPEA: diisopropylethylamine
• NBS: N-bromosuccinimide
• NIS: N-iodosuccinimide
• TsCl: tosyl chloride
• MeCN: acetonitrile
• MeOH: methanol
• MS: mass spectrometry
• ESI: Electrospray ionization
• m -CPBA: metachloroperbenzoic acid
• EtOH: ethanol
• Moz-ON: (2- (4-Methoxybenzyloxycarbonyloxyimino) -2-phenylacetonitrile)
• Boc-OSu: N- ( tert- butoxycarbonyloxy) succinimide
• TFA: trifluoro acetic acid
• Ph: phenyl
• TMS = trimethylsilyl
• TSTU: O- (N-Succinimidyl) -1,1,3,3-tetramethyluronium tetrafluoroborate
• DCC: dicyclohexylurea
• PtF: melting point
• δ: chemical shift
• ppm: parts per million
• Hz: Hertz
• min: minute
• h: hour
• cAMP: cyclic adenosyl monophosphate
• Mops: 3- (N-morpholino) propanesulfonic acid
• HTRF: Homogeneous Fluorescence in Solved Time

The general strategy for synthesizing chromophores involves a Sonogashira coupling that has been widely described in the literature ( Sonogashira et al Tetrahedron Letters, 50 (1975) 4467-4470 , Rossi et al Organic Preparation and International Procedure 27 (1995) 129-160 . This reaction makes it possible to couple a true alkyne with an aromatic halide (preferably an iodinated or brominated derivative) or a tosyl. True alkynes ( 8a and 8b ) are not commercially available. They were prepared according to the synthesis described in scheme 1 and detailed in the experimental part. Thus, the polyethylene glycol derivative 1 has been activated in the form of a tosylated compound. Protection with a Boc protecting group of amine 3 was necessary to avoid polyalkylation. The two alkylations of iodophenol led to the two phenolic ethers 6a and 6b with suitable yields of 51 and 75%, respectively. Couplings of Sonogashira using trimethylsilylacetylene followed by deprotection of the TMS group to the two desired true alkynes 8a and 8b.

Synthesis of chromophores "carboxylates" is described in Scheme 2. The chélidamique acid 9 is converted to diester 10 chlorinated as has been described by Maury et al (Inorganic Chemistry 50 (2011) 4987-4999 ). The chlorine-iodine exchange is carried out in the presence of sodium iodide under ultrasound to lead to the compound 11. The key step of this synthesis is to selectively reduce the diester 11 in the presence of sodium borohydride at 0 ° C to obtain mono-alcohol monoester with a yield of 60%. Finally, the chromophore backbone is obtained using a second Sonogashira reaction thus leading to the compounds 13a and 13b. Finally, the activation of the alcohols was carried out via a mesylation reaction.

The preparation of "phosphinate" chromophores is described in Scheme 3. The objective of the synthesis is to provide a derivative of orthogonally trisubstituted pyridine. Thus commercially available 2-bromo-6-methylpyridine is oxidized to N-oxide analog 16 simply by oxidation in the presence of metachloroperbenzoic acid (m-CPBA). The activation of this nitrogenous heterocycle makes it possible to easily introduce the nitro group at the 4-position corresponding to the third functional group. The N-oxide is then removed using the phosphorus tribromide leading to the free pyridinyl derivative. The introduction of the phosphinate group was carried out using a palladium coupling in the presence of commercial phenylphosphinic acid. As regards the pyridine methylphosphinate, it is obtained by coupling of the ethylmethyl phosphinate derivative, itself obtained by hydrolysis of diethylmethyl phosphonite using 1 equivalent of water. The development of these couplings proved to be particularly difficult and finally made it possible to obtain the desired compounds 19a and 19b using microwaves as a source of energy but also by thermal means. The following description of the synthesis was carried out for example on the series of phenyl phosphinates but the results are extrapolable to methyl phosphinates since these two types of compounds have identical reactivities. Substitution of the nitro group by a bromine atom was carried out in the presence of acetyl bromide. Although this reagent is effective in this reaction, it causes the formation of hydrobromic acid which hydrolyzes the phosphinate group. This is reintroduced immediately after using ethyl orthoformate. Functionalization of methyl at the 6-position of pyridine is carried out using a rearrangement described by Ginsburg et al (American Chemical Society Journal 79 (1957) 481-485 ). The pyridine is oxidized a second time using the same conditions described previously. This N-oxide function is sufficiently nucleophilic to react with acetic anhydride which undergoes an intermediary rearrangement thereby introducing an acetate group at the 6-position leading to the key intermediates of the synthesis 23a and 23b. The phosphinate chromophore backbone is elaborated using the Sonogashira reaction in the presence of the true alkynes previously described. The ester (acetate) is hydrolysed to the corresponding alcohol which is converted to the brominated derivative leading to the phosphinate activated chromophores 26a-f. Alternatively it is also possible to prepare the corresponding mesylated derivatives as previously described for the "carboxylate" chromophores thus leading to the 26g-I derivatives .

Regarding asymmetric azamacrocycles (series 34 ) bearing or without an X functional group, they were prepared by condensation of the diol derivative with a dinosyl compound 31 using Mitsunobu conditions. The deprotection of the nosyl groups is carried out conventionally in the presence of thioglycolic acid and potassium carbonate. This reaction sequence makes it possible, for example, to obtain all the variations of the substituted or unsubstituted azamacrocycles (147TACN, 159TACD, 148TACD and 148TACU).

A first example of a non-functionalized phosphinate complex is described in scheme 5. The azamacrocycle is condensed on the brominated phosphinate chromophore in the presence of potassium carbonate. The phosphinate esters are hydrolysed with potassium or lithium hydroxide leading to the ligands which are then used to form the europium 37a-c complexes .

The europium complexes are prepared according to schemes 6 and 7. From the mono-substituted macrocycles 147TACN and 159TACD, two "chromophore carboxylate or phosphinate" OPEG "units are condensed leading to the 42a-b or 47a-c derivatives . In terms of yield, comparable results were obtained with brominated or mesylated phosphinate chromophores. The protecting group Boc is deprotected in the presence of trifluoroacetic acid followed by the alkylation of the third chromophore having a masked NH ₂ group, useful for the conjugation to a biomolecule. The three ester functions (carboxylate or phosphinate) are then hydrolysed and the Boc group is removed in acidic medium. The formation of the lanthanide complex is carried out by reacting the ligands 41 and 46 with the corresponding lanthanide salts and in this case the chloride or the europium acetate.

The methodology for preparing the hybrid complexes is described in both schemes 9 and 10. It consists in using synthons prepared previously in the context of the synthesis of phosphinate or carboxylate complexes.

From the dialkyl derivatives (dicarboxylate -OPEG or diphosphinate -OPEG) are condensed the mesylated phosphinate derivatives (for the dicarboxylates) or the mesylated carboxylate derivatives (for the diphosphinates). These reactions lead to the hybrid ligands which are then hydrolysed and complexed with the rare earth and in particular with europium using europium chloride or acetate.

The functionalized NH ₂ complex is not usable in the state to be bioconjugated to a biomolecule: proteins, antibodies, peptides, sugars, etc. To carry out these bioconjugations, it is necessary to convert this NH ₂ group into a biocompatible functional group. . Below is described the methodology:

For the maleimide complexes, they are directly prepared using a bifunctional commercial reagent: N- [β-maleimidopropyloxy] succinimidyl, which leads to the desired functionalization. With regard to obtaining the functionalized complexes in the form of NHS ester, it is necessary to convert the amine function to the carboxylic acid derivative (condensation of glutaric anhydride on the amino group) beforehand and then to activate this function as an NHS ester using either TSTU or the conventional DCC, NHS conditions.

The condensation of the complexes on the M2BG-Glu-NHS derivative, the synthesis of which is described in the patent application WO2010034931 , leads to conjugated complexes that can be used in a labeling reaction of the Snap protein.

The condensation of the complexes on the cAMP derivative makes it possible to obtain a whole series of complexes conjugated to cAMP. These complexes all have the same photophysical properties and can be used in the HTRF tests developed by Cisbio Bioassays.

The nature of the W group depends on the chromophore to be synthesized and has no influence on chromophore synthesis.

Regarding the acetylenic acetylated derivative 71, it was obtained by simple acetylation reaction in the presence of acetic anhydride using the corresponding commercial precursor.

Chromophores that are not commercially available were simply prepared using method A or B.

Method A is broken down into three stages: halogenation of the commercial aromatic derivative followed by coupling of Sonogashira with trimethylsilylacetylene leading to derivative 76, deprotection of the trimethylsilyl protecting group can be carried out in situ or not during the second reaction of Sonogashira which allows to obtain the chromophore 78. According to the method B, the reaction sequence is carried out in the reverse order, namely: Sonogashira reaction on the iodopyridine derivative 12 leading to the derivative 77 which is deprotected in situ during the second coupling thus leading to the chromophore 78.

The general methodology for obtaining symmetrical complexes is described in scheme 14: the chromophores are mesylated then introduced on the nitrogenous macrocycle (here for example triazacyclononane). Hydrolysis of the three ester functions to carboxylic acids and complexation with europium leads to the corresponding complex. This approach quickly discriminates a number of complexes having the desired photophysical properties and avoids the more tedious preparation of functionalized dissymmetric complexes. Indeed, it has been determined that the photophysical properties of a symmetric europium complex (TACN or TACD complex) (three identical chromophores) were the same as those of an asymmetric europium complex (two identical chromophores and a chromophore bearing a -LG group allowing the conjugation with a biomolecule ).

EXPERIMENTAL PART General informations chromatography

Thin-layer chromatography was performed on Merck 60 F ₂₅₄ silica gel plates on aluminum foil or on Merck 60 F ₂₅₄ (E-type) aluminum foil plates on aluminum foil.

• HPLC Analytique : ThermoScientific, pompe quaternaire P4000, Détecteur UV 1000 à lampe au deutérium (190-350 nm), colonne analytique Waters XBridge C18, 3.5 µm, 4.6 × 100 mm.
• HPLC Préparative : Shimadzu, 2 pompes LC-8A, détecteur UV à barrette de diodes Varian ProStar, colonne préparative Waters XBridge prép. C18, 5 µm: 19 × 100 mm ou 50 × 150mm.

High performance liquid chromatography (HPLC) analytical and preparative were performed on two devices:

• Analytical HPLC: ThermoScientific, P4000 quaternary pump, UV detector 1000 with deuterium lamp (190-350 nm), Waters XBridge C18 analytical column, 3.5 μm, 4.6 × 100 mm.
• Preparative HPLC: Shimadzu, 2 LC-8A pumps, Varian ProStar UV diode array detector, Waters XBridge preparative column prep. C18, 5 μm: 19 × 100 mm or 50 × 150 mm.

The silica column chromatographies were carried out on Merck silica gel 60 (0.040-0.063 mm). The alumina column chromatographies were performed on Sigma-Aldrich aluminum oxide, neutral, activated, Brochmann I.

spectroscopy at. Nuclear magnetic resonance

• s : singulet, se : singulet élargi, d : doublet, t : triplet, q : quadruplet, m : multiplet, dd : doublet dédoublé, td : triplet dédoublé, qd : quadruplet dédoublé, ddd : doublet de doublet dédoublé.

The NMR spectra ( ¹ H, ¹³ C and ³¹ P) were carried out using a Bruker Avance 400 MHz NanoBay spectrometer (9.4 Tesla magnet), equipped with a BBFO measurement probe, multi-nuclei. diameter 5 mm, gradient Z and lock ² H. The chemical shifts (δ) are expressed in parts per million (ppm). The following abbreviations are used:

• s: singlet, se: widened singlet, d: doublet, t: triplet, q: quadruplet, m: multiplet, dd: doublet split, td: doubled triplet, qd: doubled quadruplet, ddd: double doublet doubled.

b. Mass spectrometry

The mass spectra (LC-MS) were performed using a Waters ZQ 2000 single-quadrupole ESI / APCI multimode source spectrometer equipped with Waters XBridge C18, 3.5μm, 4.6 × 100mm column.

vs. High resolution mass spectrometry

The analyzes were performed with a QStar Elite (Applied Biosystems SCIEX) mass spectrometer equipped with a pneumatically assisted atmospheric pressure ionization (API) source. The sample was ionized in positive electrospray mode under the following conditions: electrospray voltage (ISV): 5500 V; orifice voltage (OR): 20 V; Nebulization gas pressure (air): 20 psi. The high resolution mass spectrum (MS) was obtained with a flight time analyzer (TOF). The exact mass measurement was done in triplicate with a double internal calibration.

Various

Melting point apparatus: The melting points were made using a BUCHI melting point B-540 apparatus.

To a solution of triethylene glycol monomethyl ether 1 (33.2 g, 0.2 mol) in tetrahydrofuran (50 mL) was added dropwise a solution of sodium hydroxide (14.56 g of sodium hydroxide in 60 g). mL of water, 0.36 mol). The reaction medium was cooled to 0 ° C and a solution of toluene-4-sulfonyl chloride in tetrahydrofuran (38.5 g in 60 mL, 0.2 mol) was added dropwise. At the end of the addition, the reaction medium was stirred at room temperature for 12 h. The progress of the reaction was monitored by TLC. After this period, the reaction was complete. The reaction medium was extracted with diethyl ether (3 x 100 mL). The organic phases were combined, dried over sodium sulphate and filtered. The solvent was removed under reduced pressure to yield compound 2 as a slightly yellow oil. The compound was sufficiently pure to be used in the subsequent synthesis without further purification (57.22 g, 89%). ¹ H NMR (200 MHz, CDCl ₃ ): δ: 7.77 (d, J = 8.3 Hz, 2H), 7.31 (d, J = 8.3 Hz, 2H), 4.13 (m , 2H), 3.57 (m, 10H), 3.33 (s, 3H), 2.41 (s, 3H).

To a solution of 3-bromopropylamine hydrobromide 3 (3.72 g, 16.9 mmol) in dichloromethane (100 mL) was added di- tert- butyl dicarbonate (3.71 g, 16.9 mmol). ) and triethylamine (10 mL). The reaction mixture was stirred at room temperature for 12 h. The progress of the reaction was monitored by TLC. After this period the reaction was complete. The solvent was removed under reduced pressure. To this residue was added a saturated solution of sodium chloride (100 mL) and the mixture was extracted with diethyl ether (2 x 50 mL). The organic phases were combined, washed with saturated sodium chloride solution (3 × 50 mL) and dried over sodium sulfate. After filtration, the solvent was removed under reduced pressure to afford compound 4 as a slightly brown solid. The compound was sufficiently pure to be used in the subsequent synthesis without further purification (3.50 g, 87%). ¹ H NMR (200 MHz, CDCl ₃ ) δ: 4.63 (s, 1H), 3.42 (t, J = 6.5 Hz, 2H), 3.26 (td, J = 6.5; 6.5 Hz, 2H), 2.03 (m, J = 6.5 Hz, 2H), 1.43 (s, 9H). HMRS (ESI) calcd for C ₈ H ₁₆ NO ₂ Br [M + H ⁺ ], m / z 255.0703, found: 255.0695.

Of 4-iodophenol A suspension of 5 (4.0 g, 18 mmol) and potassium carbonate (12.4 g, 90 mmol) in dimethylformamide (50 mL) was added the tosyl derivative 2 (8.7 g 27 mmol). The reaction mixture was refluxed for 36 h. The progress of the reaction was monitored by TLC. After this period the reaction was complete. The reaction mixture was cooled to room temperature and sintered. The filtrate was concentrated under reduced pressure and the residue was purified by silica column chromatography (cyclohexane / ethyl acetate 95/5 up to 50/50 in 5% increments) to yield compound 6a as a a yellow oil (3.38 g, 51%). ¹ H NMR (200 MHz, CDCl ₃ ) δ: 7.52 (d, J = 9.0 Hz, 2H), 6.68 (d, J = 9.0 Hz, 2H), 4.07 (t, J = 6.0 Hz, 2H), 3.82 (t, J = 5.2 Hz, 2H), 3.62 (m, 6H), 3.54 (m, 2H), 3.36 (s, 3H). ¹³ C NMR (50 MHz, CDCl ₃ ) δ 158.7; 138.2; 117.1; 82.9; 71.9; 70.9; 70.7; 70.6; 69.6; 67.6; 50.1. HMRS (ESI) calculated for C ₁₃ H ₁₉ O ₄ I [M + H ^+], m / z 384.0666, found: 384.0668.

A of 4-iodophenol solution of 5 (3.00 g, 13.6 mmol) in anhydrous acetonitrile (100 mL) was added sodium carbonate (5.65 g, 40.9 mmol). The reaction was refluxed for 1 hour then to this suspension was added the brominated derivative 4 (2.60 g, 10.9 mmol). The reaction mixture was refluxed for 12 h. The progress of the reaction was monitored by TLC. After this period the reaction was complete. The reaction mixture was cooled to room temperature and the solvent was removed under reduced pressure. To this residue was added water (100 mL) and the mixture was extracted with dichloromethane (2 x 50 mL). The organic phases were combined, dried over sodium sulphate and filtered. The solvent was removed under reduced pressure and the residue was purified by silica column chromatography (dichloromethane) to give compound 6b as a white solid (3.10 g, 75%). PtF: 79-80 ° C. ¹ H NMR (500 MHz, CDCl ₃ ) δ: 7.52 (d, J = 8.9 Hz, 2H), 6.64 (d, J = 8.9 Hz, 2H), 4.69 (s, 1H), 3.95 (t, J = 6.2 Hz, 2H), 3.29 (td, J = 6.2, 6.2 Hz, 2H), 1.94 (m, J = 6, 2 Hz, 2H), 1.41 (s, 9H), ¹³ C NMR (125 MHz, CDCl ₃ ) δ: 158.7; 155.9; 138.2; 116.9; 82.9; 65.9; 37.9. HMRS (ESI) calcd for C ₁₄ H ₂₀ NO ₃ I [M + H ⁺ ], m / z 378.0561, found: 378.0559.

A solution of iodine compound 6a (1.00 g, 2.7 mmol) in a mixture of tetrahydrofuran (20 mL) and triethylamine (20 mL) was degassed with stirring for 20 min. To this solution were added trimethylsilylacetylene (0.8 g, 8.2 mmol) followed by palladium (II) bis-triphenylphosphine bis-chloride (19 mg, 0.02 mmol) and copper iodide ( I) (10 mg, 0.054 mmol). The reaction mixture was stirred at room temperature for 12 h. The progress of the reaction was monitored by TLC. After this period, the reaction was complete. The solvents were removed under reduced pressure. To the residue was added a solution of saturated ammonium chloride (50 mL) and the mixture was extracted with dichloromethane (2 x 25 mL). The organic phases were combined and washed with saturated ammonium chloride solution (50 mL) and then with sodium chloride solution (2 × 50 mL) and finally dried over sodium sulfate. After filtration, the solvent was removed under reduced pressure and the residue was purified by chromatography on silica (dichloromethane / ethyl acetate 95/5 90/10) to give compound 7a in the form of a slightly yellow oil (0, 64 g, 70%). ¹ H NMR (200 MHz, CDCl ₃ ) δ: 7.36 (d, J = 8.8 Hz, 2H), 6.82 (d, J = 8.8 Hz, 2H), 4.11 (t, J = 5.8 Hz, 2H), 3.83 (t, J = 5.1 Hz, 2H), 3.64 (m, 6H), 3.53 (m, 2H), 3.34 (s, 3H); ), 0.24 (s, 9H). ¹³ C NMR (50 MHz, CDCl ₃ )?: 159.0; 133.3; 115.4; 114.4; 105.2; 92.3; 71.9; 70.8; 70.6; 70.5; 69.6; 67.4; 58.9; 0.1. HMRS (ESI) calcd for C ₁₈ H ₂₈ O ₄ Si [M + H ⁺ ], m / z 337.1830, found: 337.1828.

A solution of iodinated derivative 6b (2.50 g, 6.6 mmol) in a mixture of tetrahydrofuran (20 mL) and triethylamine (20 mL) was degassed with stirring for 20 min. To this solution were added trimethylsilylacetylene (1.95 g, 19.8 mmol) followed by bis-triphenylphosphine bis-chloride of palladium (II) (46 mg, 0.06 mmol) and copper iodide (I ) (25 mg, 0.13 mmol). The reaction was stirred at room temperature for 12 h. The progress of the reaction was monitored by TLC. After this period the reaction was complete. The solvents were removed under reduced pressure. To the residue was added a solution of saturated ammonium chloride (50 mL) and the mixture was extracted with dichloromethane (2 x 25 mL). The organic phases were combined and washed with a saturated ammonium chloride solution (50 ml) and then with a solution of saturated sodium chloride (2 × 50 ml) and then dried over sodium sulfate. After filtration, the solvent was removed under reduced pressure and the residue was purified by chromatography on a silica column (dichloromethane / pentane 1/1 then dichloromethane) to give compound 7b in the form of a white solid (1.50 g , 65%). PtF: 92-93 ° C. ¹ H NMR (200 MHz, CDCl ₃ ) δ: 7.37 (d, J = 8.7 Hz, 2H), 6.78 (d, J = 8.7 Hz, 2H), 4.69 (s, 1H), 3.99 (t, J = 6.2 Hz, 2H), 3.29 (td, J = 6.2 Hz and J = 6.2 Hz, 2H), 1.95 (m, J = 6.2 Hz, 2H), 1.42 (s, 9H), 0.22 (s, 9H). ¹³ C NMR (100 MHz, CDCl ₃ )?: 159.08; 156.13; 133.61; 115.54; 114.44; 105.28; 92.63; 79.41; 65.92; 38.06; 29.66; 28.54; 0.20. HMRS (ESI) calcd for C ₁₉ H ₂₉ NO ₃ Si [M + H ⁺ ], m / z 348.1989, found: 348.1993.

To a solution of silylated derivative 7a (5.7 g, 17 mmol) in a mixture of tetrahydrofuran (60 mL) and methanol (60 mL) was added potassium carbonate (7.0 g, 58 mmol). The reaction medium was stirred at room temperature for 2 h. The progress of the reaction was monitored by TLC. After this period the reaction was complete. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by chromatography on a silica column (dichloromethane / ethyl acetate 95/5 then 90/10) to give compound 8a in the form of a yellow oil (3.9 g, 87%). ¹ H NMR (200 MHz, CDCl ₃ ) δ: 7.40 (d, J = 8.8 Hz, 2H), 6.84 (d, J = 8.8 Hz, 2H), 4.12 (t, J = 5.1 Hz, 2H), 3.84 (t, J = 4.5 Hz, 2H), 3.64 (m, 6H), 3.54 (m, 2H), 3.36 (s, 3H), 2.98 (s, 1H). ¹³ C NMR (50 MHz, CDCl ₃ ) δ: 159.4; 133.7; 114.8; 114.5; 83.9; 76.5; 72.1; 71.0; 70.8; 70.7; 69.8; 67.7; 59.1.

To a solution of silylated derivative 7b (1.27 g, 3.7 mmol) in a mixture of tetrahydrofuran (30 mL) and methanol (30 mL) was added potassium carbonate (1.52 g, 11.0 g). mmol). The reaction medium was stirred at room temperature for 2 h. The progress of the reaction was monitored by TLC. After this period the reaction was complete. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by silica column chromatography (pentane / dichloromethane 50/50 to 100% in 10% increments) to afford compound 8b as a white solid (0.86 g, 85%) . ¹ H NMR (200 MHz, CDCl ₃ ) δ: 7.39 (d, J = 8.7 Hz, 2H), 6.81 (d, J = 8.7 Hz, 2H), 4.69 (s, 1H), 3.99 (t, J = 6.2 Hz, 2H), 3.29 (td, J = 6.2 Hz and J = 6.2 Hz, 2H), 2.97 (s, 1H). ), 1.95 (m, J = 6.2 Hz, 2H), 1.42 (s, 9H).

To a solution of chelidamic acid 9 (17 g, 93 mmol) in thionyl chloride (95 mL) were added a few drops of dimethylformamide. This solution was heated at 100 ° C for 48 hours. Thionyl chloride was removed under reduced pressure. At this residue a dichloromethane (50 mL) was added and the mixture was cooled to 0 ° C. To this mixture was added dropwise over a period of 10 min anhydrous methanol (40 mL) and the mixture was allowed to warm to room temperature overnight. The solvents were removed under reduced pressure and to this residue was added a saturated solution of sodium bicarbonate (100 mL). The mixture was filtered and the filtrate was extracted with dichloromethane (3 x 50 mL), the organic phases were pooled, dried over sodium sultate, filtered and evaporated under reduced pressure to give a white solid identified as the compound 10 . The product was sufficiently pure to be used in the further synthesis without further purification (13.3 g, 63%). ¹ H NMR (300 MHz, CDCl ₃ ) δ: 8.32 (s, 2H), 4.06 (s, 6H).

To a solution of compound 10 (6.0 g, 26.2 mmol) in anhydrous acetonitrile (140 mL) was added sodium iodide (39 g, 262 mmol) and the mixture was sonicated for 20 min. To this mixture was added acetyl chloride (5.55 mL, 78.6 mmol) and the mixture was sonicated for 5 h. To this solution cooled to 0 ° C was added a saturated solution of sodium bicarbonate (75 mL) followed by addition of water (100 mL). The mixture was extracted with ethyl acetate (2 × 50 mL), and the organic phases were combined, washed with 0.2 M thiosufalt solution, and finally dried over sodium sulfate, filtered and concentrated. under reduced pressure. To this residue was added methanol (40 mL), the mixture was stirred for 20 min and then filtered to give a white solid identified as compound 11 . The product was sufficiently pure to be used in the further synthesis without further purification (6.9 g, 82%). ¹ H NMR (300 MHz, CDCl ₃ ) δ: 8.68 (s, 2H), 4.05 (s, 6H).

To a solution of diester 11 (7.0 g, 21.8 mmol) in dichloromethane (50 mL) cooled to 0 ° C was added methanol (35 mL) followed by the addition of sodium borohydride (540 mg). 14.2 mmol). The mixture was stirred for 2h at 0 ° C and then a solution of 1M hydrochloric acid (20mL) was added. The solvent was removed under reduced pressure and the residue A saturated solution of sodium bicarbonate (100 mL) was added and the mixture was extracted with ethyl acetate (4 x 30 mL). The organic phases were combined, dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica column chromatography (60/40 cyclohexane / ethyl acetate to 0/100 in 10% increments) to give compound 12 as a white solid (3.8 g, 60%). ¹ H NMR (300 MHz, CDCl ₃ ) δ: 8.41 (s, 1H), 7.99 (s, 1H), 4.85 (s, 2H), 4.02 (s, 3H). ¹³ C NMR (75 MHz, CDCl ₃ ) δ: 164.44; 161.44; 147.31; 133.34; 133.06; 106.97; 64.31; 53.29.

A solution of acetylene derivative 8a (3.9 g, 15 mmol) and iodine derivative 12 (2.8 g, 9.6 mmol) in a mixture of tetrahydrofuran (20 mL) and triethylamine (20 mL) was degassed. with stirring for 20 min. To this solution was added palladium (II) bis-triphenylphosphine bis (104 mg, 0.15 mmol) and copper (I) iodide (56 mg, 0.29 mmol). The reaction was stirred at room temperature for 12 h. The progress of the reaction was monitored by TLC. After this period, the reaction was complete. The solvents were removed under reduced pressure. To the residue was added a solution of saturated ammonium chloride (50 mL) and the mixture was extracted with dichloromethane (2 x 25 mL). The organic phases were combined, washed with saturated ammonium chloride solution (50 mL) and then with saturated sodium chloride solution (2 × 50 mL) and then dried over sodium sulfate. After filtration, the solvent was removed under reduced pressure and the residue was purified by silica column chromatography (dichloromethane / methanol 1/1) to yield product 13a as a yellow solid (3.2 g, 78%). %). PtF: 47 ° C. ¹ H NMR (500 MHz, CDCl ₃ ) δ: 8.04 (s, 1H), 7.56 (s, 1H), 7.45 (d, J = 8.8 Hz, 2H), 6, 89 (d, J = 8.8 Hz, 2H), 4.82 (s, 2H), 4.13 (m, 2H), 3.97 (s, 3H), 3.84 (m, 3H), 3.71 (m, 2H), 3.64 (m, 4H), 3.52 (m, 2H), 3.35 (s, 3H), 1.89 (s, 1H). ¹³ C NMR (50 MHz, CDCl ₃ ) δ: 165.4; 160.7; 160.1; 147.3; 134.1; 133.8; 125.8; 125.5; 115.1; 114.1; 95.9; 85.4; 72.1. HMRS (ESI) calcd for C ₂₃ H ₂₇ NO ₇ [M + H ⁺ ], m / z 430.1860, found: 430.1858.

A solution of acetylenic derivative 8b (0.864 g, 3.1 mmol) and iodine derivative 12 (0.735 g, 2.5 mmol) in a mixture of tetrahydrofuran (20 mL) and triethylamine (20 mL) was degassed with stirring. for 20 minutes. To this solution was added bis-triphenylphosphine bis-chloride palladium (II) (22 mg, 0.031 mmol) and copper iodide (I) (12 mg, 0.063 mmol). The reaction was stirred at room temperature for 12 h. The progress of the reaction was monitored by TLC. After this period the reaction was complete. The solvents were removed under reduced pressure. To the residue was added a solution of saturated ammonium chloride (50 mL) and the mixture was extracted with dichloromethane (2 x 25 mL). The organic phases were combined, washed with saturated ammonium chloride solution (50 mL) and then with saturated sodium chloride solution (2 × 50 mL) and then dried over sodium sulfate. After filtration the solvent was removed under reduced pressure and the residue was purified by silica column chromatography (98/2 dichloromethane / methanol) to give compound 13b as a white solid (0.910 g, 82%). PtF 143-144 ° C. ¹ H NMR (500 MHz, CDCl ₃ ) δ: 8.04 (s, 1H), 7.58 (s, 1H), 7.45 (d, J = 8.7 Hz, 2H), 6, 86 (d, J = 8.7 Hz, 2H), 4.83 (d, J = 5.4 Hz, 2H), 4.75 (s, 1H), 4.01 (t, J = 6, 1 Hz, 2H), 3.97 (s, 3H), 3.31 (td, J = 6.1, 6.1 Hz, 2H), 1.96 (m, J = 6.1 Hz, 2H). , 1.41 (s, 9H). ¹³ C NMR (125 MHz, CDCl ₃ ) δ: 165.4; 160.8; 160.1; 156.2; 147.3; 134.1; 133.8; 125.8; 125.4; 114.9; 113.9; 95.9; 85.4; 66.1; 64.8; 53.2; 38.1; 29.7; 28.6. HMRS (ESI) calcd for C ₂₄ H ₂₈ N ₂ O ₆ [M + H ⁺ ], m / z 441.2020, found: 441.2021.

To a cooled solution at 5 ° C of alcohol 13a (300 mg, 0.7 mmol) in tetrahydrofuran (20 mL) was added triethylamine (212 mg, 0.21 mmol) followed by mesyl chloride (88 mg). mg, 0.77 mmol). The reaction medium was stirred at 0 ° C for 30 min and then at room temperature for 12 h. The progress of the reaction was monitored by TLC. After this period, the reaction was complete. The solvent was removed under reduced pressure. To the residue was added water (30 mL) and the mixture was extracted with dichloromethane (2 x 30 mL). The organic phases were combined, washed with water (30 mL) and dried over sodium sulfate. After filtration the solvent was removed under reduced pressure and the residue was purified by column chromatography on silica (dichloromethane / ethyl acetate 8/2) to give compound 14a as a slightly yellow solid (330 mg, 93%). ¹ H NMR (500 MHz, CDCl ₃ ) δ: 8.09 (s, 1H), 7.66 (s, 1H), 7.45 (d, J = 8.7 Hz, 2H), 6, 88 (d, J = 8.7 Hz, 2H), 5.37 (s, 2H), 4.12 (m, 2H), 3.97 (s, 3H), 3.83 (m, 2H), 3.69 (m, 2H), 3.62 (m, 4H), 3.51 (m, 2H), 3.33 (s, 3H), 3.13 (s, 3H), ¹³ C-NMR (50Hz, m.p. MHz, CDCl ₃ ) δ: 164.9; 160.1; 154.6; 147.8; 134.6; 133.7; 126.6; 126.3; 114.9; 113.6; 96.8; 84.9; 71.9; 70.9; 70.7; 70.6; 69.6; 67.6; 59.1; 53.1; 38.1. HMRS (ESI) calcd for C ₂₄ H ₂₉ NO ₉ S [M + H ⁺ ], m / z 508.1636, found: 508.1637.

To an alcohol solution 13b (21.9 mg, 50 μmol) in anhydrous tetrahydrofuran (1 mL) was added dropwise triethylamine (18.1 mg, 150 μmol) under an inert atmosphere. To this cooled mixture at 4 ° C. was added dropwise a solution of mesyl chloride (6 μL, 75 μmol) in anhydrous tetrahydrofuran (0.2 mL). The progress of the reaction was monitored by TLC. After 20 min, the reaction was complete. The solvent was removed under reduced pressure. The residue was dissolved in dichloromethane (4 mL) and this solution was washed with water (3 × 5 mL). The organic phase was dried over magnesium sulfate, filtered and concentrated under reduced pressure to yield a yellow-green oil (26.9 mg, quantitative). Product 14b was sufficiently pure to be used in the subsequent synthesis without further purification.

To a solution of 2-bromo-6-methylpyridine 15 (30 g, 174 mmol) in chloroform (300 mL) was added at room temperature m -chloroperbenzoïque acid (62 g, 261 mmol). The mixture was heated at 65 ° C for 20 h and then cooled at 0 ° C for 3 h. After filtration of the precipitate, the filtrate was concentrated under reduced pressure. To this residue was added an aqueous solution of sodium hydroxide (2N, 75 mL) and this solution was extracted with dicloromethane (3 x 100 mL). The organics were pooled, dried over sodium sulfate, filtered and concentrated under reduced pressure to afford compound 16 as a yellow solid used in the subsequent synthesis without further purification (15 g, 67%); PtF: 48-55 ° C; ¹ H NMR (400 MHz, CDCl ₃ ) δ: 7.55 (dd, J = 7.9 Hz, 1.6 Hz, 1H), 7.23 (dd, J = 7.9 Hz, 1.6). Hz, 1H), 7.07 (t, J = 7.9 Hz, 1H), 2.57 (s, 3H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ: 151.2; 133.5; 128.7; 125.3; 125.2; 19.3. HMRS (ESI) calcd for C ₆ H ₇ NOBr [M + H ⁺ ], m / z 187.9711, found: 187.9698.

To a solution of compound 16 (10 g, 53 mmol) in concentrated sulfuric acid (14.1 mL, 265 mmol) cooled to 0 ° C was added dropwise fuming nitric acid (10.4 g). mL, 252 mmol). The mixture was stirred at room temperature for 15 minutes and was then heated at 85 ° C for 16 hours. The solution was then cooled to room temperature and poured into crushed ice (60 g). After stirring for 15 minutes, the precipitate was filtered and washed with water. The yellow solid was dissolved in dichloromethane and the solution was dried over sodium sulfate, filtered and concentrated under reduced pressure to give compound 17 in the form of a yellow solid which was used in the course of the synthesis without purification additional. PtF 137-138 ° C.

To a solution of N-oxide compound 17 (11.7 g, 50 mmol) in chloroform (300 mL) was added phosphorus tribromide (14.2 mL, 150 mmol) and the mixture was heated to 60.degree. ° C under argon for 16 h. The solution was cooled to room temperature and concentrated under reduced pressure and then poured into ice-cold 2M sodium hydroxide solution (260 mL). The mixture was extracted with dichloromethane (3 x 100 mL), the organic phases were combined, dried over sodium sulfate and concentrated under reduced pressure. The resulting oil was purified by silica column chromatography using dichloromethane as eluent thereby yielding compound 18 as a yellow solid (8.4 g, 78%). PtF 51-52 ° C.

To a solution of 2-bromo-6-methyl-4-nitropyridine 18 (1.01 g, 4.68 mmol) in dry toluene (10 mL) was added phenylphosphinate (0.95 g, 5.60 mmol) and triethylamine (2.6 mL, 19.0 mmol). The mixture was degassed by three cycles of freeze-thaw. To this solution was added tetrakis (triphenylphosphine) palladium (0) (83 mg, 0.07 mmol) and the mixture was again degassed three times before being stirred under reflux for 16 h under argon. The progress of the reaction was monitored by TLC. After this period, the reaction was complete. The solution was cooled and diluted with dichloromethane (20 mL). The mixture was washed with aqueous hydrochloric acid solution (1M, 2 × 15 mL) followed by washing with water (3 × 15 mL). The organic phase was dried over sodium carbonate potassium, filtered and the solvent was removed under reduced pressure to obtain a dark residue which was purified by column chromatography on silica (0.5% dichloromethane / methanol) thus giving compound 19a in the form of a yellow oil ( 645 mg, 45%). ¹ H NMR (400 MHz, CDCl ₃ ) δ: 8.55 (dd, J = 5.6 Hz, J = 1.4 Hz, 1H), 7.97 (ddd, J = 11.2 Hz, 7, 7 Hz, 1.4 Hz, 2H), 7.90 (d, J = 1.4 Hz, 1 H), 7.55 (td, J = 7.7 Hz, 1.4 Hz, 1 H), 7.46 (td, J = 7.7 Hz, 3.5 Hz, 2H), 4.15 (qd, J = 7.0 Hz, 4.2 Hz, 2H), 2.72 (s, 3H) 1.38 (t, J = 7.0 Hz, 3H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ: 163.0 (d, J = 21 Hz), 158.1 (d, J = 167 Hz), 154.0 (d, J = 13 Hz), 132, 9 (d, J = 3 Hz), 132.5 (d, J = 10 Hz), 129.1 (d, J = 140 Hz), 128.5 (d, J = 13 Hz), d, J = 24Hz), 117.5 (d, J = 3Hz), 62.2 (d, J = 6Hz), 24.9, 16.4; ³¹ P NMR (162 MHz, CDCl ₃ ) δ: 23.7. HMRS (ESI) calcd for C ₁₄ H ₁₆ N ₂ O ₄ P [M + H ⁺ ], m / z 307.0848, found: 307.0851.

To diethylmethyl phosphonite (2 g, 14.7 mmol) was added at 0 ° C water (264 μL, 14.7 mmol) without any particular precautions. The reaction mixture was warmed to room temperature over 1 h and then stirred for 16 h. To this mixture was added a solution of 2-bromo-6-methyl-4-nitropyridine 18 (2.1 g, 12.3 mmol) in dry toluene (20 mL), and triethylamine (6 mL, 43 mL). 0 mmol). The mixture was degassed by three cycles of freeze-thaw. To this solution was added tetrakis (triphenylphosphine) palladium (0) (320 mg, 0.27 mmol) and the mixture was again degassed three times before being stirred under reflux for 16 h under argon. The progress of the reaction was monitored by TLC. After this period, the reaction was complete. The solution was cooled and diluted with dichloromethane (20 mL). The mixture was washed with aqueous hydrochloric acid solution (1M, 2 × 15 mL) followed by washing with water (3 × 15 mL). The organic phase was dried over potassium carbonate, filtered and the solvent was removed under reduced pressure to obtain a dark residue which was purified by column chromatography on silica (dichloromethane / methanol 1.6% with an increment of 0 , 1%) thus giving compound 19b as a colorless oil (700 mg, 29%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.75 (dd, J = 7.0 Hz ³ J _HP 10 Hz, 1H), 7.59 (td, ³ J = 7.0 Hz J = 4, 8 Hz, 1H), 7.16 (d, J = 7.0 Hz, 1H), 3.86 (dqd, J = 80 Hz, J = 7.0 Hz, J = 4.5 Hz, 2H), 2.50 (s, 3H), 1.66 (d, J = 15 Hz, 3H), 1.61 (t, J = 7.0 Hz, 3H); δ _C (CDCl ₃ ) 159.4 (d, J 20 Hz), 153.7 (d, J = 158 Hz), 136.0 (d, J = 10 Hz), 125.6 (d, J = 3 Hz), 124.6 (d, J = 22Hz), 60.8 (d, J = 6Hz), 24.5, 16.3, 13.3 (d, J = 103 Hz); δ _P (CDCl ₃ ) +41.2; m / z HMRS (ESI) calcd for C ₉ H ₁₄ NO ₂ P [M + H ⁺ ], m / z 200.0858, found: 200.0862.

To the nitro derivative 19a (2.00 g, 6.54 mmol) was added acetyl bromide (15 mL, 0.2 mol) and the mixture was stirred at 70 ° C. for 16 h under argon. During this period a pale brown precipitate had formed. The precipitate and solution were cautiously added dropwise into methanol (100 mL) cooled to 0 ° C. The solvent was removed under reduced pressure to yield the desired compound as a pale brown solid which was used directly in the subsequent synthesis without purification (1.81 g, 90%); ¹ H NMR (400 MHz, CD ₃ OD) δ: 8.33 (dd, J = 7.2 Hz, 2.0 Hz, 1H), 8.23 (d, J = 2.0 Hz, 1H). ), 7.95 (ddd, J = 13.2 Hz, 7.6 Hz, 1.6 Hz, 2H), 7.63 (1H, td, J = 7.6 Hz, 1.6 Hz, 1 H), 7.55 (2H, td, J = 7.6Hz, 3.6Hz), 2.77 (3H, s); ¹³ C NMR (100 MHz, CD ₃ OD): δ 159.4 (d, J = 20 Hz), 151.7 (d, J = 160 Hz), 145.1 (d, J = 10 Hz), 134 , 8 (d, J = 3 Hz), 133.3 (d, J = 10 Hz), 131.0 (d, J = 24 Hz), 130.6 (d, J = 3 Hz), 130.2 (d, J = 140 Hz), 129.6 (d, J = 12 Hz), 20.4; ³¹ P NMR (162 MHz, CD ₃ OD) δ: 14.3. HMRS (ESI) calcd for C ₁₂ H ₁₀ NO ₂ P [M + H ⁺ ], m / z 309.9633, found: 309.9648.

To phosphinic acid 20a (1.80 g, 5.80 mmol) was added ethyl orthoformate (50 mL) and the mixture was stirred at 140 ° C for 72 h under argon. The progress of the reaction was monitored by TLC. After this period, the reaction was complete. The solution was cooled to room temperature and the solvent was removed under reduced pressure. The residue was purified by column chromatography on silica (0.5% dichloromethane / methanol) to give compound 21a as a yellow oil (1.08 g, 55%); ¹ H NMR (400 MHz, CDCl ₃ ) δ: 8.04 (dd, J = 6.3 Hz, 1.4 Hz, 1H), 7.95 (ddd, J = 11.2 Hz, 7.0 Hz). , 1.4 Hz, 2H), 7.51 (1H, td, 7.0Hz, 1.4Hz, 1H), 7.43 (td, J = 7.0Hz, 3.5Hz, 2H) ), 7.37 (d, J = 1.4 Hz, 1H), 4.11 (qd, J = 7.0 Hz, 4.2 Hz, 2H), 2.52 (s, 3H), 1 , 34 (t, J = 7.0 Hz, 3H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ: 161.2 (d, J = 22 Hz), 155.7 (d, J = 165 Hz), 133.5 (d, J = 15 Hz), 132.7 (d, J = 3 Hz), 132.6 (d, J = 22 Hz), , J = 10 Hz), 130.0 (d, J = 139 Hz), 128.5 (d, J = 3 Hz), 128.4 (d, J = 23 Hz), 128.3 (d, J) = 13 Hz), 62.1 (d, J = 6 Hz), 24.5, 16.7; ³¹ P NMR (162 MHz, CDCl ₃ ) δ 25.5. HMRS (ESI) calcd for C ₁₄ H ₁₆ NO ₂ BrP [M + H ⁺ ], m / z 340.0102, found: 340.0102.

To a solution of pyridinyl derivative 21a (1.25 g, 3.68 mmol) in chloroform (20 mL) was added meta-chloroperbenzoic acid (1.27 g, 7.35 mmol). The resulting solution was stirred at 65 ° C for 16 h under argon. The progress of the reaction was monitored by TLC. After this period, the reaction was complete. The mixture was cooled to room temperature and the solvent was removed under reduced pressure to give a yellow oil. This oil was dissolved in dichloromethane and was washed with sodium bicarbonate solution (0.5M, 50 mL). The phases were separated and the aqueous phase was extracted with dichloromethane (3 × 30 mL). The organic phases were combined, dried over magnesium sulfate and the solvent was removed under reduced pressure. The resulting yellow oil was purified by silica column chromatography (0-2% dichloromethane / methanol in 0.1% increments) to obtain a yellow oil corresponding to compound 22a (1.11 g, 75%). ¹ H NMR (400 MHz, CDCl ₃ ) δ: 8.05 (dd, J = 7.7 Hz, 2.1 Hz, 1H), 7.98 (dd, J = 7.7 Hz, 13.3 Hz) , 2H), 7.50 (t, 7.7 Hz, 1H), 7.44 (d, J = 2.1 Hz, 1H), 7.41 (td, J = 7.7 Hz, 4.2 Hz, 2H), 4.13 (qd, J = 5.6 Hz, 4.9 Hz, 2H), 2.32 (s, 3H), 1.34 (t, J = 5.6 Hz, 3H). ; ¹³ C NMR (100 MHz, CDCl ₃ ) δ: 151.0 (d, J = 4 Hz), 144.2 (d, J = 149 Hz), 133.2 (d, J = 11 Hz), 133, 1 (d, J = 4 Hz), 133.0 (d, J = 11 Hz), 132.2 (d, J = 4 Hz), 129.0 (d, J = 152 Hz), d, J = 14Hz), 117.4 (d, J = 12Hz), 62.3 (d, J = 6Hz), 17.5, 16.7; ³¹ P NMR (162 MHz, CDCl ₃ ) δ: +21.2. HMRS (ESI) calcd for C ₁₄ H ₁₆ O ₃ BrNP [M + H ⁺ ], m / z 356.0051, found: 356.0061.

To a solution of methylpyridinyl derivative 21a (1.18 g, 3.47 mmol) in carbon tetrachloride (50 mL) were added N-bromosuccinimide (0.92 g, 5.21 mmol) and then benzoyl (30 mg, 1.2 mmol). The mixture was refluxed under an inert atmosphere and under irradiation using a 100 W lamp for 16 hours. The progress of the reaction was monitored by ¹ H NMR. After this period, the reaction was complete. The reaction mixture was cooled to room temperature and the solvent was removed under reduced pressure and the residue was solubilized in dichloromethane (30 mL) and then washed with potassium carbonate solution (25 mL). The organic phase was dried over magnesium sulfate, filtered and the solvent was removed under reduced pressure. The crude product was purified by silica column chromatography (cyclohexane / 0% ethyl acetate to 30% in increments of 2%) to afford compound 22c as a colorless oil (466 mg, 32%). %). ¹ H NMR (400 MHz, CDCl ₃ ) δ: 8.16 (dd, J = 6.0 Hz, J = 1.6 Hz, 1H), 7.96 (ddd, J = 12.4 Hz, J = 6, 8 Hz, J = 1.6 Hz, 2H), 7.69 (d, J = 1.6 Hz, 1H), 7.53 (td, J = 6.8 Hz, J = 1.6 Hz, 1H), 7.44 (td, J = 6.8 Hz J = 3.6 Hz, 2H), 4.49 (s, 2H), 4.12 (qd, J = 6.8 Hz, J = 4.2 Hz, 2H), 1.36 (t, J = 6.8 Hz, 3H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ: 159.2 ( J = 21 Hz), 156.1 (d, J = 163 Hz), 134.5 ( J = 14 Hz), 133.0 ( J = 3 Hz), 132.7 ( J = 10 Hz), 130.8 ( J = 23 Hz), 129.4 ( J = 139 Hz), 128.7 ( J = 3 Hz), 128.6 ( J = 13 Hz), 62.4 (d, J = 6 Hz), 32.5, 16.7; ³¹ P NMR (162 MHz, CDCl ₃ ) δ: 24.9; HMRS (ESI) calcd for C ₁₄ H ₁₅ NO ₂ PBr ₂ [M + H ⁺ ], m / z 417.9207, found: 417.9212.

To the pyridinyl derivative N-Oxide 22a (1.8 g, 5.1 mmol) was added acetic anhydride (35 mL) and the solution was heated at 120 ° C for 3 h with stirring. The progress of the reaction was monitored by ³¹ P NMR. After this period, the reaction was complete. The solution was cooled to room temperature and the solvent was removed under reduced pressure. The residue was purified by silica column chromatography (0-1% dichloromethane / methanol in 0.1% increments) to give a yellow oil corresponding to 23a (0.66 g, 33%); ¹ H NMR (400 MHz, CDCl ₃ ) δ: 8.16 (dd, J = 7.7 Hz, 2.1 Hz, 1H), 7.93 (dd, J = 7.7 Hz, 13.3 Hz) , 7.5 (d, J = 2.1 Hz, 1H), 7.51 (t, J = 7.7 Hz, 1H), 7.43 (td, J = 7.7 Hz, , 2 Hz, 2H), 5.18 (s, 2H), 4.09 (qd, J = 5.6 Hz, 4.9 Hz, 2H), 2.12 (s, 3H), 1.33 (s, 2H), t, J = 5.6 Hz, 3H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ: 170.5, 158.6 (d, J = 4 Hz), 156.0 (d, J = 149 Hz), 134.3 (d, J = 25 Hz) ), 132.9 (d, J = 5 Hz), 132.7 (d, J = 10 Hz), 130.5 (d, J = 23 Hz), 129.5 (d, J = 139 Hz), 128.6 (d, J = 13Hz), 126.6 (d, J = 3Hz), 66.0, 62.2 (d, J = 6Hz), 21.0, 16.7; ³¹ P NMR (162 MHz, CDCl ₃ ) δ: +25.0. HMRS (ESI) calcd for C ₁₆ H ₁₈ O ₄ BrNP [M + H ⁺ ], m / z 398.0151, found: 398.0157.

To the brominated derivative 23a (540 mg, 1.36 mmol) was added anhydrous tetrahydrofuran (8 mL) and the solution was degassed by three cycles of freeze-thaw. To this solution was added acetylenic derivative 8a (456 mg, 1.36 mmol) followed by triethylamine (4 mL) and the solution was degassed again. To this solution was added tetrakis (triphenylphosphine) palladium (0) (156 mg, 0.136 mmol) and copper iodide (26 mg, 0.136 mmol). This new solution was again degassed three times and was stirred under argon and immediately after, to this mixture was added a solution of tetrabutylammonium fluoride (1M in tetrahydrofuran, 1.9 mL, 2.04 mmol). A color change from yellow to dark blue was observed and the mixture was stirred at 65 ° C under argon. The progress of the reaction was monitored by TLC. After 3 hours the reaction was complete. The reaction mixture was cooled and the solvent was removed under reduced pressure and the crude product was purified by silica column chromatography (dichloromethane / methanol 0 to 1.5% in 0.1% increments) to obtain an oil. yellow corresponding to compound 24a (575 mg, 73%). ¹ H NMR (400 MHz, CDCl ₃ ) δ: 8.05 (dd, J = 6.0 Hz, 1.6 Hz, 1H), 7.96 (dd, J = 8.4 Hz, 12.4 Hz , 2H), 7.50 (t, J = 8.4 Hz, 1H), 7.44 (d, J = 8.8 Hz, 2H), 7.43 (2H, td, J = 8.4). Hz, 4.2 Hz, 2H), 7.42 (d, J = 1.6 Hz, 1H), 6.88 (d, J = 8.8 Hz, 2H), 5.21 (s, 2H) ), 4.12 (qd, J = 5.6 Hz, 4.8 Hz, 2H), 4.11 (t, J = 4.8 Hz, 2H), 3.83 (t, J = 4.8). Hz, 2H), 3.71 (t, J = 4.8 Hz, 2H), 3.65 (t, J = 4.8 Hz, 2H), 3.63 (t, J = 4.8 Hz, 2H), 3.61 (t, J = 4.8 Hz, 2H), 3.34 (s, 3H), 2.13 (s, 3H), 1.34 (t, J = 5.6 Hz, 3H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ: 170.6; 160.1; 157.3 (d, J = 20 Hz); 154.7 (d, J = 165 Hz), 133.8; 133.2 (d, J = 12 Hz), 132.7 (d, J = 5 Hz), 132.6 (d, J = 10 Hz), 130.1 (d, J = 139 Hz), 128, Δ (d, J = 22 Hz), 128.6 (d, J = 12 Hz), 124.6 (d, J = 3 Hz), 115.1; 114.1; 96.2; 85.5 (d, J = 2Hz), 72.2; 71.1; 70.9; 70.8; 69.8; 67.8; 66.5; 62.1 (d, J = 6Hz), 59.3; 21.1; 16.7; ³¹ P NMR (162 MHz, CDCl ₃ ) δ: +26.0. HMRS (ESI) calcd for C ₃₁ H ₃₇ O ₈ NP [M + H ⁺ ], m / z 582.2257, found: 582.2260.

To the brominated derivative 23a (170 mg, 0.43 mmol) was added anhydrous tetrahydrofuran (3 mL) and the solution was degassed by three cycles of freeze-thaw. To this solution was added acetylenic derivative 8b (148 mg, 0.43 mmol) and triethylamine (1.5 mL) and the solution was degassed again. To this solution was added tetrakis (triphenylphosphine) palladium (0) (49 mg, 0.043 mmol) and copper iodide (8 mg, 0.043 mmol). This new solution was again degassed three times and was stirred under argon and immediately after, to this mixture, was added a solution of tetrabutylammonium fluoride (1 M in THF, 0.6 mL, 0.64 mmol) . A color change from yellow to dark blue was observed and the mixture was stirred at 65 ° C under argon. The progress of the reaction was monitored by TLC. After 3 hours the reaction was complete. The reaction mixture was cooled to room temperature and the solvent was removed under reduced pressure. The crude product was purified by silica column chromatography (dichloromethane / methanol 0-1.5% in increments of 0.1%) to give a yellow oil corresponding to compound 24b (174 mg, 69%); ¹ H NMR (400 MHz, CDCl ₃ ) δ: 8.09 (dd, J = 6.0 Hz, 2.0 Hz, 1H), 7.99 (dd, J = 8.4 Hz, J = 12.4). Hz, 2H), 7.54 (t, J = 8.4 Hz, 1H), 7.47 (d, J = 8.4 Hz, 2H), 7.45 (td, J = 8.4 Hz). , 4.2 Hz, 2H), 7.45 (d, J 2.0 Hz, 1H), 6.88 (d, J = 8.4 Hz, 2H), 5.24 (s, 2H), 4 , 75 (se, 1H), 4.14 (qd, J = 5.6 Hz, 4.8 Hz, 2H), 4.05 (t, J = 6 Hz, 2H), 3.33 (m, 2H) ), 2.17 (s, 3H), 1.92 (q, J = 6Hz, 2H), 1.44 (s, 9H), 1.38 (t, J = 5.6Hz, 3H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ: 170.7; 160.0; 157.3 (d, J = 20.3 Hz), 156.2; 154.6 (d, J = 167 Hz), 133.9; 133.2 (d, J = 12 Hz), 132.7 (d, J = 5 Hz), 132.6 (d, J = 10 Hz), 130.1 (d, J = 138 Hz), 129, 1 (d, J = 23Hz), 128.6 (d, J = 13Hz), 124.6 (d, J = 3 Hz), 114.9; 114.1; 96.2; 85.5 (d, J = 2Hz), 79.5; 66.5; 66.1; 62.1 (d, J = 6Hz), 38.1; 29.7; 28.6; 21.1; 16.7; ³¹ P NMR (162 MHz, CDCl ₃ ) δ: + 26.1. HMRS (ESI) calcd for C ₃₂ H ₃₈ O ₇ N ₂ P [M + H ⁺ ], m / z 593.2417 found: 593.2435.

Compounds (24c), (24d), (24e), (24f)

These compounds were obtained following the procedure used for compounds 24a and 24b.

To a solution of the acetylated derivative 24a (50 mg, 0.086 mmol) in absolute ethanol (2.5 mL) was added a catalytic amount of sodium metal (~ 2 mg) and the solution was heated with stirring for 40 min under inert atmosphere. The progress of the reaction was monitored by TLC. After this period, the reaction was complete. To this mixture was added dichloromethane (25 mL) and the sodium salts were then filtered through silica. The silica was rinsed with 10% dichloromethane / ethanol (300 mL). The solvent was removed under reduced pressure and the crude product was purified by silica column chromatography (dichloromethane / methanol 0 to 2.4% in increments of 0.2%) to obtain a colorless oil corresponding to compound 25a (40%). mg, 87%). ¹ H NMR (400 MHz, CDCl ₃ ) δ: 8.06 (dd, J = 6.0 Hz, 1.6 Hz, 1H), 7.95 (dd, J = 8.4 Hz, 12.4); Hz, 2H), 7.53 (t, J = 8.4 Hz, 1H), 7.44 (d, J = 8.8 Hz, 2H), 7.43 (td, J = 8.4 Hz). 4.2 Hz, 2H), 7.39 (d, J = 1.6 Hz, 1H), 6.90 (d, J = 8.8 Hz), 4.75 (s, 2H), 4 , 14 (qd, J = 5.6 Hz 4.8 Hz, 2H), 4.11 (t, J = 4.8 Hz, 2H), 3.86 (t, J = 4.8 Hz, 2H) , 3.73 (t, J = 4.8 Hz, 2H), 3.68 (t, J = 4.8 Hz, 2H), 3.66 (t, J = 4.8 Hz, 2H), 3 , 63 (t, J = 4.8 Hz, 2H), 3.37 (s, 3H), 1.37 (t, J = 5.6 Hz, 3H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ: 160.4 (d, J = 19 Hz), 159.9; 154.0 (d, J = 165 Hz), 133.6, 133.0 (d, J = 12 Hz), 132.6 (d, J = 5 Hz), 132.3 (d, J = 10Hz ), 129.7 (d, J = 128 Hz), 128.6 (d, J = 22 Hz), 128.4 (d, J = 12 Hz), 123.8 (d, J = 3 Hz), 114.9, 113.8, 96.0, 85.3 (d, J = 2 Hz), 71.9, 70.9, 70.7, 70.6, 69.6, 67.5, 63, 9.61.8 (d, J = 6Hz), 59.1, 16.5; ³¹ P NMR (162 MHz, CDCl ₃ ) δ: + 26.6. HMRS (ESI) calcd for C ₂₄ H ₂₉ NO ₉ S [M + H ⁺ ], m / z 540.2151 found: 540.2142.

To a solution of the acetyl derivative 24b (32 mg, 0.054 mmol) in absolute ethanol (2.5 mL) was added a catalytic amount of sodium metal (~ 2 mg) and the solution was heated with stirring for 40 minutes. min under inert atmosphere. The progress of the reaction was monitored by TLC. After this period, the reaction was complete. To this mixture was added dichloromethane (25 mL) and the sodium salts were then filtered through silica. The silica was rinsed with 10% dichloromethane / ethanol (300 mL). The solvent was removed under reduced pressure and the crude product was purified by silica column chromatography (0-2% dichloromethane / methanol in 0.2% increments) to obtain a colorless oil corresponding to compound 25b (24 mg, 80%). ¹ H NMR (400 MHz, CDCl ₃ ) δ: 8.01 (dd, J = 6.4 Hz, 2.0 Hz, 1H), 7.89 (dd, J = 8.4 Hz, 12.4). Hz, 2H), 7.48 (t, J = 8.4 Hz, 1H), 7.40 (d, J = 8.8 Hz, 2H), 7.39 (td, J = 8.4 Hz). , 4.2 Hz, 2H), 7.33 (d, J = 2.0 Hz, 1H), 6.81 (d, J = 8.8 Hz, 2H), 4.73 (s, 1H). ), 4.69 (s, 2H), 4.08 (qd, J = 5.6 Hz, 4.8 Hz, 2H), 3.97 (t, J = 6 Hz, 2H), 3.26 ( m, 2H), 1.92 (q, J = 6 Hz, 2H), 1.37 (s, 9H, 1.31 (t, J = 5.6 Hz, 3H), ¹³ C NMR (100 MHz, m.p. CDCl ₃ ) δ: 160.3 (d, J = 18 Hz), 159.8, 156.0, 153.2 (d, J = 164 Hz), 133.7, 133.0 (d, J = 11); Hz) 132.6 (d, J = 5 Hz) 132.3 (d, J = 10 Hz) 129.6 (d, J = 138 Hz) 128.6 (d, J = 18 Hz) 128.5 (d, J = 9 Hz), 123.8 (d, J = 3 Hz), 114.7, 113.8, 96.0, 85.3 (d, J = 2 Hz); , 3, 65.9, 63.8, 61.9 (d, J = 6 Hz), 37.9, 29.5, 28.4, 16.6, ³¹ P NMR (162 MHz, CDCl ₃ ) δ HMRS (ESI) calcd for C ₃₀ H ₃₆ O ₆ N ₂ P [M + H ⁺ ], m / z 551.2311 found: 551.2290.

Compounds (25c), (25d), (25e), (25f)

These compounds were obtained following the procedure used for Examples 25a and 25b.

To a solution of the alcohol 25a (116 mg, 0.22 mmol) in anhydrous dichloromethane (5 mL) cooled to 0 ° C was added phosphorus tribromide (30 μL, 0.33 mmol). The solution was stirred for 1 hour at 0 ° C and then allowed to warm to room temperature. The progress of the reaction was monitored by TLC. After 1 h the reaction was complete. The reaction mixture was immediately purified by silica column chromatography (dichloromethane / methanol, 1%) to afford the desired compound 26a as a yellow oil (95 mg, 73%); ¹ H NMR (400 MHz, CDCl ₃ ) δ: 8.00 (dd, J = 6.0 Hz, 1.2 Hz, 1H), 7.94 (dd, J = 8.8 Hz, 12.4). Hz, 2H), 7.51 (d, J = 1.2 Hz, 1H), 7.48 (t, J = 8.8 Hz, 1H), 7.41 (d, J = 8.8 Hz, 2H), 7.40 (td, J = 8.8 Hz, 4.2 Hz, 2H), 6.85 (d, J = 8.8 Hz, 2H), 4.48 (s, 2H). , 4.09 (qd, J = 7.2 Hz, 4.8 Hz, 2H), 4.08 (t, J 4.8 Hz, 2H), 3.81 (t, J = 4.8 Hz, 2H), 3.67 (t, J = 4.8 Hz, 2H), 3.63 (t, J = 4.8 Hz, 2H), 3.59 (t, J = 4.8 Hz, 2H). , 3.49 (t, J = 4.8 Hz), 3.31 (s, 3H), 1.32 (t, J = 7.2 Hz, 3H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ: 160.1 (d, J = 19 Hz); 159.9; 154.3 (d, J = 165 Hz); 133.6; 133.3 (d, J = 12 Hz); 132.4 (d, J = 5 Hz); 132.3 (d, J = 10 Hz); 129.7 (d, J = 128 Hz); 128.6 (d, J = 22Hz); 128.4 (d, J = 13H); 126.7 (d, J = 3 Hz); 114.9; 113.8; 96.3; 84.9 (d, J = 2 Hz); 71.9; 70.9; 70.7; 70.6; 69.6; 67.5; 61.0 (d, J = 5 Hz); 59.1; 33.1; 16.6; ³¹ P NMR (162 MHz, CDCl ₃ ) δ: +24.8; HMRS (ESI) calcd for C ₂₉ H ₃₄ O ₆ NPBr [M + H ⁺ ], m / z 602.1302, found: 602.1302.

To a solution of alcohol 25b (25 mg, 0.045 mmol) in anhydrous dichloromethane (3 mL) cooled to 0 ° C was added phosphorus tribromide (30 μl, 0.33 mmol). The solution The mixture was stirred for 45 minutes at 0 ° C. and then allowed to come to room temperature. The progress of the reaction was monitored by TLC. After 30 minutes the reaction was complete. The reaction mixture was immediately purified by silica column chromatography (1% dichloromethane / methanol) to afford the desired compound 26b as a yellow oil (13 mg, 47%); ¹ H NMR (400 MHz, CDCl ₃ ) δ: 8.07 (dd, J = 6.0 Hz, 1.2 Hz, 1 H), 8.01 (dd, J = 8.4 Hz, J = 12 Hz) , 2H), 7.58 (d, J = 1.2 Hz, 1H), 7.55 (t, J = 8.4 Hz, 1H), 7.47 (td, J = 8.4 Hz) , 4.2 Hz, 2H), 7.46 (d, J = 8.8 Hz, 2H), 6.89 (d, J = 8.8 Hz, 2H), 4.72 (s, 1H) , 4.55 (s, 2H), 4.15 (qd, J = 6.0 Hz, 4.8 Hz, 2H), 4.05 (t, J = 6 Hz, 2H), 3.33 (m , 2H), 2.00 (q, J = 6Hz, 2H), 1.44 (s, 9H), 1.38 (t, J = 6.0Hz, 3H); ³¹ P NMR (162 MHz, CDCl ₃ ) δ: +25.8. HMRS (ESI) calculated for C ₃₀ H ₃₄ O ₅ N ₂ PBr [M + H ⁺ ], m / z 613.1482, found: 613.1462.

To a solution of alcohol 25a (360 mg 0.66 mmol) in anhydrous tetrahydrofuran (4 mL) cooled to 5 ° C were added triethylamine (0.3 mL, 1.98 mmol) and then mesyl chloride. (77 μL, 0.99 mmol) under an inert atmosphere. The reaction medium was stirred at ambient temperature for 15 minutes. The progress of the reaction was monitored by TLC. After this period, the reaction was complete. The solvent was removed under reduced pressure. To the residue was added water saturated with sodium chloride (30 mL) and the mixture was extracted with dichloromethane (2 x 30 mL). The organic phases were combined and dried over magnesium sulfate. After filtration, the solvent was removed under reduced pressure to yield a yellowish oil corresponding to compound 26g (430 mg, 99%) which was sufficiently pure to be used in the next step without further purification.

To a solution of alcohol 25b (118 mg 0.21 mmol) in anhydrous tetrahydrofuran (7 mL) cooled to 5 ° C was added triethylamine (87 μL, 0.64 mmol) followed by mesyl chloride (24 mL). μL, 0.32 mmol) under an inert atmosphere. The reaction medium was stirred at ambient temperature for 15 minutes. The progress of the reaction was monitored by TLC. After this period, the reaction was complete. The solvent was removed under reduced pressure. To the residue was added water saturated with sodium chloride (30 mL) and the mixture was extracted with dichloromethane (2 x 30 mL). The organic phases were combined and dried over magnesium sulfate. After filtration, the solvent was removed under reduced pressure to yield a brown solid corresponding to 26h (125 mg, 95%) which was sufficiently pure for use in the next step without further purification.

Compounds (26i), (26j), (26k), (26I)

These compounds were obtained following the procedure used for Examples 26g and 26h.

To a solution of 1,4,7-triazacyclononane hydrochloride 27a (188 mg, 0.78 mmol) in dioxane / water (10 mL, 8: 2), was added 2- (4-methoxybenzyloxycarbonyloxyimino) -2-phenylacetonitrile (477 mg, 1.5 mmol). The reaction mixture was homogenized by vigorous stirring and then a solution of triethylamine (540 μL, 3.8 mmol) in dioxane / water (10 mL, 8: 2) was added. The progress of the reaction was monitored by TLC. After 24 h, the reaction was complete. The solvent was removed under reduced pressure. The residue was dissolved in dichloromethane. This solution was washed with water saturated with sodium chloride (3 × 10 mL). The organic phase was dried over magnesium sulfate, filtered and concentrated under reduced pressure. The crude was purified by alumina column chromatography (dichloromethane / methanol 98: 2 to 90:10 in increments of 1%) to afford the desired compound 28a as an oil (266 mg, 74%). ). ¹ H NMR (400 MHz, CDCl ₃ ) δ: 7.32-7.25 (m, 4H), 6.88-6.85 (m, 4H), 5.09 (m, 4H), 3.80 (s, 6H), 3.56-3.47 (m, 4H), 3.31-3.25 (m, 4H), 2.87 (m, 4H). ¹³ C NMR (100 MHz, CDCl ₃ ) δ: 159.68; 156.76; 130.05; 128.92; 114.03; 67.15; 55.41; 52.20; 51.98; 51.26; 50.31; 50.07; 49.19; 48.18; 47.73; 47.66.

To a solution of 1,4,7-triazacyclododecane 27b (1 g, 5.84 mmol) in dioxane (50 mL), was added 2- (4-methoxybenzyloxycarbonyloxyimino) -2-phenylacetonitrile (3.62 g, 11.7 mmol). The reaction mixture was homogenized by vigorous stirring and was stirred at room temperature under an inert atmosphere for 2 h. The progress of the reaction was monitored by TLC. After this period, the reaction was complete. The solvent was removed under reduced pressure. The residue was dissolved in dichloromethane (50 mL). This solution was washed with water saturated with sodium chloride (2 × 20 mL). The aqueous phases were combined and extracted with dichloromethane (25 mL). The organic phases were combined, dried over magnesium sulphate, filtered and concentrated under reduced pressure. The crude was purified by alumina column chromatography (dichloromethane / methanol 100: 0 to 97: 3 in increments of 0.5%) to afford the desired compound 28b as an oil (980 mg. 34%). HMRS (ESI) calcd for C ₂₇ H ₃₇ N ₃ O ₆ [M + H ⁺ ], m / z 500.2761, found: 500.2758.

To a solution of diprotected macrocycle 28a (50 mg, 0.1 mmol) in dichloromethane (7 mL) was added N- ( tert- butoxycarbonyloxy) succinimide (36 mg, 0.15 mmol). The solution was stirred at room temperature under an inert atmosphere. The progress of the reaction was monitored by TLC. After 24 h, the reaction was complete. The solution was washed directly with water saturated with sodium chloride (3 × 10 mL). The organic phase was dried over magnesium sulfate, filtered and concentrated under reduced pressure. The crude was purified by column chromatography on alumina (90:10 dichloromethane / methanol to 70:30 in 5% increments) to yield the desired compound 29a as a brown oil (44 mg. , 72%). ¹ H NMR (400 MHz, CDCl ₃ ) δ: 7.30-7.19 (m, 4H), 6.88-6.83 (m, 4H), 5.06-4.92 (m, 4H) , 3.77 (s, 6H), 3.46-3.39 (m, 12H), 1.55 (s, 9H). ¹³ C NMR (100 MHz, CDCl ₃ ) δ: 159.65; 156.56; 155.76; 130.04; 128.98; 114.00; 80.01; 67.15; 55.38; 49.74; 49.64; 49.47; 49.37; 49.12; 48.92; 48.80; 28.53; 27.75; 25.60. HMRS (ESI) calcd for C ₂₉ H ₃₉ N ₃ O ₈ [M + H ⁺ ], m / z 558.2815, found: 558.2808.

To a diMoz 28b macrocycle solution (980 mg, 1.96 mmol) in dichloromethane (40 mL) was added N- ( tert- butoxycarbonyloxy) succinimide (630 mg, 2.93 mmol). The reaction mixture was stirred at room temperature for 24 h. The progress of the reaction was monitored by TLC. After this period, the reaction was complete. This solution was washed with water saturated with sodium chloride (2 × 20 mL). The aqueous phases were combined and extracted with dichloromethane (2 × 30 mL). The organic phases were combined, dried over magnesium sulphate, filtered and concentrated under reduced pressure. The crude was purified by column chromatography on alumina (cyclohexane / ethyl acetate 100: 0 until 70:30 in 5% increments) to yield the desired compound 29b as an oil (507 mg). , 42%).

To diMoZ-monoBoc macrocycle solution 29a (300 mg, 0.5 mmol) in methanol (25 mL) was added charcoal-absorbed palladium hydroxide (about 100 mg). The reaction medium was placed in a hydrogenator and was maintained under vigorous stirring under a hydrogen atmosphere (3.45 bars - 50 PSI). The progress of the reaction was monitored by TLC alumina. After one night, the reaction was complete. The reaction medium was filtered through celite and then concentrated under reduced pressure. The colorless oil obtained was dissolved in methanol (3 mL). To this solution was added an aqueous solution of 1M hydrochloric acid (pH 2-3). The mixture was concentrated under reduced pressure and redissolved in a minimum of methanol (2 mL). To this solution was added diethyl ether (35 mL). The white precipitate obtained was collected by filtration to give a white solid corresponding to hydrochloride 30a (102 mg, 62%). PtF: 172-174 ° C. ¹ H NMR (500 MHz, D ₂ O) δ: 3.74 (t, J = 4.5 Hz, 4H), 3.63 (s, 4H), 3.48 (t, J = 4.5 Hz , 4H), 1.47 (s, 9H). ¹³ C NMR (125 MHz, CDCl ₃ ) δ: 136.0; 62.9; 25.9; 22.2; 21.7; 6.7. HMRS (ESI) calcd for C ₁₁ H ₂₃ N ₃ O ₂ [M + H ⁺ ], m / z 230.1869, found: 230.1863.

To a diMoZ-monoBoc 29b macrocycle solution (507 mg, 0.846 mmol) in methanol (25 mL) was added charcoal-absorbed palladium hydroxide (about 100 mg). The reaction medium was placed in a hydrogenator and was maintained under vigorous stirring under a hydrogen atmosphere (3.45 bars - 50 PSI). The progress of the reaction was monitored by LC-MS. After one night, the reaction was complete. The reaction medium was filtered through celite and then concentrated under reduced pressure. The colorless oil obtained was dissolved in methanol (2 mL) To this solution was added an aqueous solution of 1M hydrochloric acid (pH 2-3). The mixture was concentrated under reduced pressure and redissolved in a minimum of methanol (1 mL). To this solution was added diethyl ether (150 mL). The resulting white precipitate was collected by sinter filtration to give a white solid corresponding to 30b hydrochloride (125 mg, 43%). PtF 149-168 ° C.

To a brominated derivative solution 26c (110 mg, 0.234 mmol) in anhydrous acetonitrile (5 mL) was added under an inert atmosphere potassium carbonate (32 mg, 0.234 mmol) and 1,4,7- triazacyclononane (10 mg, 0.078 mmol). The reaction medium was refluxed for 16 hours. The progress of the reaction was monitored by TLC. After this period, the reaction was complete. The reaction medium was cooled to room temperature and the solvent was removed under reduced pressure. To the residue was added water (10 mL) and the mixture was extracted with ethyl acetate (2 x 20 mL). The organic phases were combined and dried over magnesium sulfate. After filtration, the solvent was removed under reduced pressure. The crude product obtained was purified by silica column chromatography (dichloromethane / methanol, 0 to 30% in increments of 1%) to yield compound 35a as a yellowish oil (23 mg, 23%). ¹ H NMR (400 MHz, CDCl ₃ ) δ: 8.03 (dd, J = 6.0 Hz, 3H), 7.94 (dd, J = 12.4 Hz, J = 6.8 Hz, 6H) , 7.58 (s, 3H), 7.45 (d, J = 8.8 Hz, 6H), 7.42 (t, J = 6.8 Hz, 3H), 7.37 (td, J = 6.8 Hz J = 3.6 Hz, 6H), 6.87 (d, J = 8.8 Hz, 6H), 4.10 (qd, J = 7.2 Hz, J = 4.2 Hz, 6H), 3.83 (s, 6H), 3.82 (s, 9H), 2.74 (s, 12H), 1.33 (t, J = 7.2 Hz, 9H); ³¹ P NMR (162 MHz, CDCl ₃ ) δ: + 26.6; HMRS (ESI) calculated for C ₇₅ H ₇₆ N ₆ O ₉ P ₃ [M + H ⁺ ], m / z 1297.4860, found: 1297.4860.

Compounds (35b) and (35c)

These compounds were obtained following the procedure used for Example 35a.

To a solution of the ligand 35a (10 mg, 7.7 μmol) in methanol (3 mL) was added an aqueous solution of sodium hydroxide (1 mL, 0.1 M). The solution was heated at 60 ° C for 16 h. The progress of the reaction was monitored by ¹ H NMR. After this period, the reaction was complete. The pH of the solution was adjusted to 7 by addition of hydrochloric acid (1M). To this solution was added europium acetate (3.4 mg, 8.5 μmol) dissolved beforehand in an H ₂ O: CH ₃ OH solution (0.5 mL, 1: 1 v / v). The reaction mixture was heated at 50 ° C for 24 h. The reaction mixture was cooled to room temperature and the solvent was removed under reduced pressure and the crude product was purified by silica column chromatography (5% dichloromethane / methanol) to give the desired compound 37a as a white solid (2.5 mg, 24%). ³¹ P NMR (162 MHz, CDCl ₃ ) δ: +17.5. HMRS (ESI) calculated for C ₆₉ H ₆₁ N ₆ O ₉ P ₃ Eu [M + H ⁺ ], m / z 1361.2910, found: 1361.2830.

Compounds (37b) and (37c)

These compounds were obtained following the procedure used for Example 37a.

To a solution of TACN monoBoc hydrochloride 30a (20.2 mg, 67 μmol) in anhydrous acetonitrile (20 mL) was added potassium carbonate (55.5 mg, 0.40 mmol) under an inert atmosphere. . To this suspension was added a solution of chromophore OPEG mesylated 14a (70 mg, 0.14 mmol) in anhydrous acetonitrile (2 mL). The progress of the reaction was monitored by analytical HPLC. After stirring and heating at 60 ° C for 2h, the reaction medium was cooled to room temperature. The solvent was removed under reduced pressure. The reaction crude was purified by silica column chromatography (98: 2 dichloromethane / methanol to 80: 20 in increments of 2%) to afford compound 38a as an oil (52.8 mg, 75%). HMRS (ESI) calcd for C ₅₇ H ₇₃ N ₅ O ₁₄ [M + H ⁺ ], m / z 1052.5232, found: 1052.5256.

Compound (38b)

This compound was obtained following the procedure used for Example 38a.

To a solution of ODP 38a monoBoc-dibras TACN (1.47 mg, 1.4 μmol) in dichloromethane (950 μL) was added trifluoroacetic acid (50 μL). The progress of the reaction was monitored by HPLC. After stirring at room temperature overnight, the solvent was removed under reduced pressure. The medium was taken up in methanol (1 mL) and toluene (3 mL) and then evaporated to remove traces of trifluoroacetic acid. The reaction medium was purified by preparative column HPLC giving compound 39a as a yellowish oil (0.75 mg, 56%). HMRS (ESI) calcd for C ₅₂ H ₆₅ N ₅ O ₁₂ [M + H ⁺ ], m / z 952.4708, found 952.4714.

Compound (39b)

This compound was obtained following the procedure used for Example 39a.

To a solution of TACN dibras OPEG 39a (4.25 mg, 4.4 μmol) in anhydrous acetonitrile (4 mL) was added sodium carbonate (1.4 mg, 1.34 μmol) under an inert atmosphere. . To this suspension was added a mesophilic NH-Boc chromophore solution 14b (3.5 mg, 6.7 μmol) in anhydrous acetonitrile (2 mL). The progress of the reaction was monitored by HPLC. After stirring and heating at 50 ° C. for 22 h, the reaction medium was cooled to ambient temperature. The solvent was removed under reduced pressure. The reaction medium was purified by preparative column HPLC. Compound 40a was obtained as a yellowish oil (1.25 mg, 21%).

To a triester methyl ligand solution 40a (1 mg, 727 nmol) in THF (150 uL) was added lithium hydroxide 1 M (50 micromol) and pure water (100 uL). To this whitish suspension was added THF (1 mL) to complete the solubilization. The reaction mixture was stirred at room temperature for 2 h. The progress of the reaction was monitored by HPLC. After this period, the reaction was complete. The solvent was removed under reduced pressure. The reaction medium was then taken up in pure water (500 μl) and acidified by the addition of a 1M aqueous hydrochloric acid solution to obtain the pH of the mixture of between 2-3. The solvent was removed under reduced pressure. The residue was purified by preparative HPLC to give the desired product as an oil (312 nmol, 43%). To the oil obtained above and a second batch (1 μmol) in dichloromethane (500 μl) was added trifluoroacetic acid (150 μl) and an additional volume of dichloromethane (2500 μl). The reaction mixture was stirred at room temperature for 10 minutes. The progress of the reaction was monitored by HPLC. After this period, the reaction was complete. The solvent was removed under reduced pressure to yield compound 41a as an oil (530 nmol, 53%) which was used in the next step without further purification.

To a solution of asymmetric tribras TACN carboxylic acid -NH ₂ 41a (16.4 μmol) in methanol (5 mL) and water (2 mL), was added sodium carbonate (10.4 mg, 98%). , 4 μmol) and europium chloride hexahydrate (16.6 mg, 32.8 μmol). The reaction mixture was heated at 50 ° C overnight. The progress of the reaction was monitored by analytical HPLC. After this period the reaction was complete. The reaction medium was cooled to room temperature and the solvent was removed under reduced pressure. The residue was purified by preparative HPLC leading to complex 42a as a colorless oil (9.15 μmol, 56%).

To a solution of 1,4,7-triazacyclononane-mono Boc 30a (22 mg, 0.096 mmol) in acetonitrile (5 mL) was added compound 26a (95 mg, 0.16 mmol) and sodium carbonate. potassium (27 mg, 0.19 mmol). The mixture was stirred under argon at 50 ° C for 1 h. The progress of the reaction was monitored by TLC. After this period, the reaction was complete. The reaction mixture was cooled to room temperature and the solvent was removed under reduced pressure. The residue was purified by silica column chromatography (dichloromethane / methanol 0-5% in increments of 0.5%) to obtain the desired compound 43a as a yellow oil (58 mg, 59%): NMR ¹ H (400 MHz, CDCl ₃ ) δ: 8.01 (m, 2H), 7.95 (m, 4H), 7.56 (s, 2H), 7.45 (m, 2H), 7.41 (d, J = 8.8 Hz, 4H), 7.40 (m, 4H), 6.87 (d, J = 8.8 Hz, 4H), 4.13 (t, J = 4.8 Hz , 4H), 4.10 (m, 4H), 3.85 (s, 4H), 3.84 (t, J = 4.8 Hz, 4H), 3.72 (t, J = 4.8 Hz). , 4H), 3.66 (t, J = 4.8 Hz, 4H), 3.63 (t, J = 4.8 Hz, 4H), 3.53 (t, J = 4.8 Hz, 4H). ), 3.35 (s, 6H), 3.24 (m, 4H), 2.97 (m, 4H), 2.53 (m, 4H), 1.45 (s, 9H), 1.33 (m, 6H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ: 161.6; 161.4 (d, J = 19 Hz); 159.7; 159.6; 155.5; 153.8; 153.7 (d, J = 165 Hz); 133.6; 133.5; 133.3; 132.4; 132.3; 130.1 (d, J = 145 Hz); 128.1; 128.0; 126.2; 125.9 (d, J = 3 Hz); 114.9; 114.7; 114.1; 114.0; 95.4; 95.1; 85.7; 85.5; 71.9; 70.8; 70.6; 70.5; 69.6; 67.5; 63.0; 49.1; 61.6; 59.0; 53.4; 28.7; 28.6; 16.5; ³¹ P NMR (162 MHz, CDCl ₃ ) δ: + 26.6, + 26.5 HMRS (ESI) calculated for C ₆₉ H ₈₈ N ₅ O ₁₄ P ₂ [M + H ⁺ ], m / z 1272.5840 found: 1272.5800.

Compound (43c)

This compound was obtained following the procedure used for Example 43a.

To a solution of 43a (47 mg, 0.037 mmol) in dry dichloromethane (1 mL) was added trifluoroacetic acid (0.5 mL). The solution was stirred under argon at 23 ° C for 30 min. The progress of the reaction was monitored by TLC. After this period the reaction was complete. The solvent was removed under reduced pressure and the residue was redissolved in dichloromethane (1 mL), which was again removed under reduced pressure. This procedure was repeated 5 times to remove excess trifluoroacetic acid. The residue was used in the subsequent synthesis without further purification. HMRS (ESI) calculated for C ₆₄ H ₈₀ N ₅ O ₁₂ P ₂ [M + H ⁺ ], m / z 1172.5270, found: 1172.5280.

Compound (44c)

This compound was obtained following the procedure used for Example 44a.

To the residue 44a obtained in the previous step dissolved in acetonitrile (4 mL) was added the brominated derivative 26b (18 mg, 0.029 mmol) and potassium carbonate (15 mg, 0.11 mmol). The suspension was stirred at 50 ° C for 30 min. The progress of the reaction was monitored by TLC. After this period the reaction was complete. The reaction mixture was cooled and the solvent was removed under reduced pressure. The residue was purified by chromatography on a silica column (dichloromethane / methanol 0 to 10% in increments of 0.5%) to give the desired compound 45a as a yellow oil (25 mg, 50%). ¹ H NMR (400 MHz, CDCl ₃ ) δ: 8.07 (d, J = 6.0 Hz, 3H), 7.85 (dd, J = 8.4, J = 12 Hz, 6H), 7, 48 (m, 3H), 7.47 (m, 3H), 7.46 (d, J = 8.8 Hz, 6H), 7.39 (m, 6H), 6.90 (d, J = 8); , 8 Hz, 6H), 4.76 (s, 1H), 4.14 (m, 6H), 4.11 (t, J = 4.8 Hz, 4H), 4.04 (t, J = 6 Hz, 2H), 3.94 (s, 6H), 3.87 (t, J = 4.8 Hz, 4H), 3.73 (t, J = 4.8 Hz, 4H), 3.68 ( t, J = 4.8 Hz, 4H), 3.65 (t, J = 4.8 Hz, 4H), 3.54 (t, J = 4.8 Hz, 4H), 3.37 (s, 6H), 3.34 (m, 2H), 2.81 (m, 12H), 1.99 (q, J = 6Hz, 2H), 1.43 (s, 9H), 1.34 (t, J = 7.2 Hz, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ: 160.8 (d, J = 19 Hz), 160.2; 156.2; 154.6 (d, J = 165 Hz); 134.0; 133.9; 132.9; 132.5 (d, J = 10 Hz); 130.2 (d, J = 138 Hz); 128.8; 128.7; 127.5; 115.1; 113.9; 97.0; 85.3; 79.6; 72.1; 71.1; 70.9; 70.8; 69.8; 67.8; 65.9; 62.0 (d, J = 6Hz); 59.3; 52.3; 53.0 to 46.0; 37.9; 29.7; 28.6; 16.8; ³¹ P NMR (162 MHz, CDCl ₃ ) δ: +26.4. HMRS (ESI) calcd for C ₉₄ H ₁₁₃ N ₇ O ₁₇ P ₃ [M + H ⁺ ], m / z 1704.7400, found: 1704.7410.

Compound (45c)

This compound was obtained following the procedure used for Example 45a.

To ligand 45a (52 mg, 30.5 μmol) were added tetrahydrofuran (6 mL) and aqueous lithium hydroxide solution (4 mL, 1 M). The solution was stirred at room temperature for 24 h. The progress of the reaction was monitored by HPLC. After this period, the reaction was complete. The reaction mixture was concentrated under reduced pressure and used in the subsequent synthesis without further purification. MS (ESI) calculated for C ₈₈ H ₁₀₃ N ₇ O ₁₇ P ₃ [M + 3H ⁺ ], m / z 1622. To a solution of the compound obtained above in dichloromethane (6 mL) was added trifluoroacetic acid (3 mL). The solution was stirred at room temperature for 2 h. The progress of the reaction was monitored by HPLC. After this period the reaction was complete. The solvent was removed under reduced pressure and the residue was redissolved in dichloromethane (6 mL). The solvent was again removed under reduced pressure. This procedure was repeated 5 times to remove excess trifluoroacetic acid. The crude product was purified by preparative HPLC to yield an oil which was used directly in the complexation step. MS (ESI) calculated for C ₈₃ H ₉₇ N ₇ O ₁₅ P ₃ [M + 3H ⁺ ], m / z 1622.

Compound (46c)

This compound was obtained following the procedure used for Example 46a.

To a solution of compound 46a (0.016 mmol) in methanol (1 mL) was added a solution of europium acetate (7 mg, 0.016 mmol) dissolved in water: methanol (0.5 mL, 1: 1). 1 v / v). The solution was stirred at 50 ° C for 14 h. The progress of the reaction was monitored by HPLC. After this period, the reaction was complete. The reaction mixture was cooled and the solvent was removed under reduced pressure. The residue was purified by silica column chromatography (CH ₂ Cl ₂ / CH ₃ OH, 10%: 1% NH ₃ in 0.1% increments) to obtain the europium complex in the form of a white solid 47a (9.1 mg, 34%); ³¹ P NMR (162 MHz, CD ₃ OD) δ: +17.5. HMRS (ESI) calculated for C ₈₃ H ₉₀ N ₇ O ₁₅ P ₃ Eu [M-CF ₃ CO ₂ ] ⁺ , m / z 1668.4900, found: 1668.4980.

Compound (47c)

This compound was obtained following the procedure used for Example 47a.

Compounds 50a, 50c, 53a and 53c

These compounds were obtained following the procedure used for Examples 47a and 47c.

To a solution of europium NH ₂ 42a complex (200 nmol) in MOPS buffer pH 7 (200 μL) was added a solution of (N- [β-maleimidopropyloxy] succinimidyl ester (41 μL, 300 nmol) in dimethylformamide prepared from a stock solution (1.92 mg N- [β-maleimidopropyloxy] succinimidyl in 1000 μl of dimethylformamide) The reaction medium was stirred at ambient temperature overnight. After this period the reaction was complete The reaction medium was purified by preparative HPLC to give compound 54a as an oil (105 nmol, 52%).

Compounds (55a) - (55c), (56a) - (56c) and (57a) - (57c)

These compounds were obtained following the procedure used for Example 54a.

To a solution of europium NH ₂ 42a complex (200 nmol) in anhydrous DMF (100 μL) was added triethylamine (1 μL). To this mixture was added a solution of glutaric anhydride (236 nmol) in dimethylformamide (27.4 μl). The mixture was stirred at room temperature overnight. The progress of the reaction was monitored by HPLC. After this period, the reaction was complete. The reaction medium was purified by preparative HPLC to yield the desired compound as an oil (62.7 nmol, 31%). ESI-MS m / z calculated for C ₇₃ H ₈₀ N ₇ O ₁₈ Eu + H ⁺ = 1382.45. To a solution of acidic complex (550 nmol) in anhydrous dimethylformamide (200 μl) were added diisopropylethylamine (0.8 μl) and then O- (N-Succinimidyl) -1,1,3,3-tetramethyluronium tetrafluoroborate (0.3 mg, 1 μmol). The reaction mixture was stirred at room temperature for 15 minutes. The progress of the reaction was monitored by HPLC. After this period, the reaction was complete. The reaction medium was purified directly by preparative HPLC to yield the NHS 58a compound (370 nmol, 74%). ESI-MS m / z calculated for C ₇₃ H ₈₀ N ₇ O ₁₈ Eu + H ⁺ = 1382.45.

Compounds (59a) - (59c), (60a) - (60c) and (61a) - (61c)

These compounds were obtained following the procedure used for Example 58a.

To a europium NH ₂ 42a complex solution (100 nmol) in dimethylformamide (50 μl) were added triethylamine (1.5 μl) and a solution of M2BG-glu-NHS (100 nmol) in dimethylformamide. (50 μL). The reaction is stirred at room temperature for 2 hours. The progress of the reaction was monitored by HPLC. After this period a second addition equivalent to the first was made (triethylamine and a solution of M2BG-glu-NHS in the dimethylformamide). The reaction was stirred at room temperature overnight. After this period, the reaction was complete. The reaction mixture was purified by preparative HPLC to yield compound 62a as an oil (68 nmol, 68%). ESI-MS m / z calculated for C ₆₈ H ₇₄ N ₇ O ₁₅ Eu + H ⁺ = 1382.45.

Compounds (63a), (63c), (63d), (64a), (64c), (64d), (65a), (65c) and (65d)

These compounds were obtained following the procedure used for Example 62a.

To a solution of europium complex -NH ₂ 42a (200 nmol) in dimethylformamide (100 μl) was added triethylamine (5 μl). A cAMP-NHS stock solution was prepared containing cAMP-NHS (200 nmol) in dimethylformamide (200 μL). This stock solution was added to the solution of the complex. The reaction medium was stirred overnight. The progress of the reaction was monitored by HPLC. The reaction medium was purified by preparative HPLC to yield compound 66a as an oil (62 nmol, 31%).

Compounds (67c), (67d), (68c), (68d), (69c) and (69d)

These compounds were obtained following the procedure used for Example 66a.

To a solution of 4-ethynylaniline 70 (2.34 g, 20 mmol) in dichloromethane (30 mL) was added acetic anhydride (2.5 mL, 40 mmol). The reaction medium was stirred at ambient temperature for 16 hours. The progress of the reaction was monitored by TLC. After this period the reaction was complete. The solvent was removed under reduced pressure. To the black color residue was added saturated sodium bicarbonate solution (15 mL) and the mixture was extracted with dichloromethane (2 x 15 mL). The organic phases were combined and then dried over magnesium sulfate. After filtration, the solvent was removed under reduced pressure. The crude product obtained was purified by flash chromatography on a silica column (ethyl acetate / cyclohexane 98/2 up to 60/40 in 5% increments) to give compound 71 in the form of a slightly pinkish solid ( 2.05 g, 64%). PtF: 118.4 to 125.3 ° C.

Compounds (72) and (73)

These compounds were obtained using the method described in the literature (Inorganic Chemistry-2005-44 (18) 6284).

Compound (74)

This compound was obtained using the method described in the literature ( Journal of the American Chemical Society 2009-131 (35) 12560 ).

To a solution of acetylenic derivative 71 (174 mg, 1.1 mmol) in anhydrous tetrahydrofuran (20 mL) and anhydrous triethylamine (10 mL) was added iodinated pyridine 12 (296 mg, 1 mmol). The mixture was degassed with stirring for 1 h. To this solution was added palladium (II) bis-triphenylphosphine bis (70 mg, 0.2 mmol) and copper (I) iodide (38 mg, 0.4 mmol). The reaction medium was stirred at 50 ° C. for 18 hours in the dark. The progress of the reaction was monitored by TLC. After this period the reaction was complete. The solvents were removed under reduced pressure. To the black residue was added water (10 mL) and the mixture was extracted with ethyl acetate (3 x 20 mL). The organic phases were combined and dried over magnesium sulfate. After filtration, The solvent was removed under reduced pressure. The crude product obtained was purified by silica column flash chromatography (0.5% dichloromethane / methanol to 20% in increments of 1%) to give compound 78a in the form of a brown solid (200 mg, 61%). %).

To the iodinated pyridine 12 (3.22 g, 11 mmol) was added anhydrous tetrahydrofuran (10 mL) and the solution was degassed by three cycles of freeze-thaw. To this solution was added trimethylsilyl acetylene (7.8 mL, 55 mmol) and diisopropylethylamine (9.6 mL, 50 mmol) and the solution was degassed again. To this solution was added tetrakis (triphenylphosphine) palladium (0) (385 mg, 0.55 mmol) and copper iodide (209 mg, 0.11 mmol). This new solution was again degassed three times and then stirred under argon. A color change from very light yellow to dark brown was observed and the mixture was stirred at 65 ° C under argon. The progress of the reaction was monitored by TLC. After 3 hours the reaction was complete. The reaction mixture was cooled to room temperature and the solvent was removed under reduced pressure. The crude product was purified by silica column chromatography (0-4% dichloromethane / methanol in 0.5% increments) to afford 77 (2.61 g, 90%); ¹ H NMR (400.13 MHz, CDCl ₃ ) δ: 8.01 (s, 1H), 7.56 (s, 1H), 4.82 (s, 2H), 3.97 (s, 3H), 0.25 (s, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ: 165.2; 160.8; 147.2; 133.4; 126.2; 126.0; 101.9; 101.3; 64.6; 53.1, 0.3. HMRS (ESI) calcd for C ₁₃ H ₁₈ O ₃ NSi [M + H ⁺ ], m / z 264.1056 found: 264.1050.

Compound (77a)

This compound was obtained using the method described in the literature on 4-nitrophenol ( Bioorganic & Medicinal Chemistry Letters 2007-17-5428 ).

To a solution of acetylenic derivative 77 (263 mg, 1 mmol) in anhydrous tetrahydrofuran (10 mL) and dry triethylamine (5 mL) was added iodized derivative 77a (370 mg, 1 mmol). The mixture was degassed with stirring for 1 h. To this solution was added a 1M solution of tetrabutylammonium fluoride in tetrahydrofuran (1.5 mL, 1.5 mmol), palladium (II) bis-triphenylphosphine bis-chloride (72 mg, 0.1 mmol) and copper (I) iodide (40 mg, 0.2 mmol). The reaction medium was stirred at 75 ° C. for 3 hours in the dark. The progress of the reaction was monitored by TLC. After this period the reaction was complete. The solvents were removed under reduced pressure. To the black residue was added water (10 mL) and the mixture was extracted with dichloromethane (3 x 20 mL). The organic phases were combined and dried over magnesium sulfate. After filtration, the solvent was removed under reduced pressure. The crude product obtained was purified by silica column flash chromatography (0.5% dichloromethane / methanol to 5% in increments of 1%) to yield compound 78u as a yellowish oil (203 mg, 54%). %).

To a solution of alcohol 78a (195 mg, 0.6 mmol) in anhydrous tetrahydrofuran (14 mL) cooled to 5 ° C were added triethylamine (0.3 mL, 1.8 mmol) and then mesyl chloride. (70 μL, 1.8 mmol) under an inert atmosphere. The reaction medium was stirred at room temperature for 15 min. The progress of the reaction was monitored by TLC. After this period, the reaction was complete. The solvent was removed under reduced pressure. To the residue was added water saturated with sodium chloride (30 mL) and the mixture was extracted with dichloromethane (2 x 30 mL). The organic phases were combined and dried over magnesium sulfate. After filtration, the solvent was removed under reduced pressure to yield a brown solid corresponding to compound 79a (231 mg, 95%) which was sufficiently pure for use in the next step without further purification.

To a solution of mesylated derivative 79a (120 mg, 0.3 mmol) in anhydrous acetonitrile (4 mL) was added under an inert atmosphere potassium carbonate (82 mg, 0.6 mmol) and 1, 4,7-triazacyclononane (12.9 mg, 0.1 mmol). The reaction medium was refluxed for 24 h. The progress of the reaction was monitored by TLC. After this period, the reaction was complete. The reaction medium was cooled to room temperature and the solvent was removed under reduced pressure. To the residue was added water (10 mL) and the mixture was extracted with ethyl acetate (2 x 20 mL). The organic phases were combined and dried over magnesium sulfate. After filtration, the solvent was removed under reduced pressure. The crude product obtained was purified by preparative HPLC to yield compound 80a as a white solid (12 mg, 12%).

This compound was obtained according to the procedure used for Example 80a .

This compound was obtained according to the procedure used for Example 80a .

This compound was obtained according to the procedure used for Example 81b below.

To a solution of ligand triester 80b (33 mg, 34 μmol) in tetrahydrofuran (3 mL) was added an aqueous solution of lithium hydroxide (1 mL, 1 M). The solution was stirred at room temperature for 30 minutes. The progress of the reaction was monitored by LC-MS. After this period the reaction was complete. The pH of the solution was adjusted to 7 by addition of hydrochloric acid (1M). To this solution was added europium chloride hexahydrate (37 mg, 102 μmol). The reaction mixture was stirred at room temperature for 30 minutes. The solvent was removed under reduced pressure and the crude product was dissolved in acetonitrile (4 mL) and purified by preparative HPLC to give the desired compound 81b as a white solid (23 mg, 63%) .

This compound was obtained according to the procedure used for Example 81b.

Example 1 Luminescence Intensity Stability of the Compounds of the Invention

The stability of the luminescence of various compounds according to the invention (37a, 47a, 53a, 62a, 66a, 67a, 69a, 81a, 81c ), as well as that of a compound of the prior art (compound 39 described by Latva et al in Journal of Luminescence, Volume 75, No. 2, September 1997, Pages 149-169 , of formula below) were measured in different media.

Solutions of each of these compounds at the concentration of 100 to 500 μM were prepared in 50 mM HEPES buffer pH 7.4 with 0.1% bovine serum albumin (BSA).
50 μl of each solution were placed in wells of 96-well plates (Corning Costar 3915), to which were added 45 μl of HEPES buffer or 50 μl of SVNN ("newborn calf serum" (supplier: Bayer)) . The wells were finally supplemented with 5μl of water (to obtain a basal signal, "base" in the figure 1 ), or 5 μl of a 400 mM solution of CaCl ₂ , MgCl ₂ , MnCl ₂ or EDTA. These solutions are known for their effect on rare earth complexes: Ca ++ and Mg ++ ions can compete with europium for binding with the complexing agent and EDTA can chelate europium. The Mn ++ ion is also known for its ability to cause luminescence quenching.
After 10 min, the luminescence was measured on a Rubystar reader at 620 nm with a measurement time of 50 μs and an integration time of 400 μs. The measured signals were normalized by the "base" signal of each compound and the results obtained are presented in Figure 1 . The error bars represent the standard deviation on three wells.
The figure 1 shows that the presence of 20 mM CaCl ₂ , MgCl ₂ , or EDTA does not significantly affect the luminescence of the compounds according to the invention, which can be correlated with the stability of these complexes.
It is noted that complexes with "phosphinate" arms such as 37a, 47a and 67a show excellent luminescence stability in the presence of MnCl ₂ compared to other complexes, including that of the prior art (Latva).
Finally, the luminescence of all the complexes according to the invention is significantly more stable than that of the complex of the prior art in the presence of SVNN, which is particularly advantageous for using these compounds for example for the analysis of blood samples in the field of diagnosis.

Example 2: Excitation Spectra of the Compounds of the Invention

The solutions of HEPES buffer compounds (pH 7.4, 0.1% BSA) of the previous example were used to obtain the excitation spectra of these compounds. For this, these solutions were placed in quartz tanks in a fluorescence spectrophotometer "Cary Eclipse" (Varian). It was verified that the absorption at 337 nm of these solutions was less than 0.05 units in a 1 cm tank.
Excitation spectra were obtained in the "phosphorescence" mode by measuring the emission at 615 nm after excitation at different wavelengths. Measurements were made with a delay of 0.1 ms and an integration time of 10 ms. The excitation slit was 5 nm, the emission slit 2.5 nm and the voltage of the photomultiplier 800 V.
The Figure 2 shows the excitation spectra obtained after normalization of the signal measured by the maximum signal for each compound. The excitation spectra of the compounds according to the invention exhibit a bathochromic shift compared with that of the compound of the prior art (Latva compound), making them particularly sensitive to excitation at 337 nm, which corresponds to the wavelength of the light emitted by nitrogen lasers, widely used in plate readers. These spectra also show that the compounds of the invention can also be excited at higher wavelengths, for example 365 nm or 380 nm. It is particularly advantageous to use these latter wavelengths for applications in microscopy, since they limit the light absorption by the biological materials present in the measuring medium and by the glassware present in the microscopes.

Example 3: Photophysical Properties of the Compounds of the Invention

Several compounds according to the invention have been prepared and their photophysical properties, in particular the wavelengths corresponding to their maximum absorption peaks (λmax), have been determined. The structures of these compounds and the results obtained are described in Table 2.

The chromophores (similar to compound 78, protected carboxylate function in ester form) were synthesized according to the protocol described in Scheme 13 using one of the two approaches "A" or "B" as described in this scheme (including for chromophores containing rings or heterocycles naphthyl, thiolanyl, imidazolyl and anthracenyl). For each chromophore, the UV spectra were recorded and the maximum wavelengths (λ _max ) of absorption were determined and reported in Table 2.

Some chromophores, europium complexes according to the invention where three chromophores are identical (symmetrical complex) were prepared using the protocol described in Scheme 14, with a TACN macrocycle (synthesis yields intermediates 79 compounds , 80 and 81 of Figure 14 are reported in Table 2). The photo-physical properties of these europium complexes have been determined and also reported in this table.

Results:

With the exception of the compound 78m, corresponding to the reference compound of the prior art (Latva compound 39 ), all the chromophores tested have a maximum absorption wavelength (λmax) of between 310 and 332 nm. It has been determined that the complexation of europium with a chromophore induces a bathochromic effect of 10 to 20 nm depending on the type of ligand structure (see λmax of chromophores 78b, 78f, 78g, 78k, 78l, and λmax corresponding europium complexes). Thus, Table 2 shows that all symmetric Europium complexes (compounds of formula 81) comprising the chromophores described in this table are very suitable for laser excitation at 337 nm. Furthermore, the europium complexes also have a sufficient quantum yield (> 10%) for these compounds to be used as fluorescent markers.

Example 4: Compared Properties

• les propriétés photophysiques d'un complexe d'europium symétrique (trois chromophores chrom₁ chrom₂ et chrom₃ identiques) sont les mêmes que celles d'un complexe d'europium dissymétrique (deux chromophores identiques et un chromophore comportant un groupe-L-G permettant le couplage avec une molécule à marquer).
• Les propriétés photophysiques des complexes d'europium phénylphosphinates (R₁=-PO(OH)-C₆H₅), méthylphosphinates (R₁= -PO(OH)CH₃)) et carboxylates (R₁= -COO^-) sont similaires.
• Les complexes d'europium hybrides dont les chromophores comportent des groupes R₁ différents, par exemple dans lesquels un des trois groupes R₁ est un groupe carboxylate et les deux autres groupes R₁ sont des groupes phénylphosphinates ou méthylphosphinates, possèdent des caractéristiques photophysiques identiques à celles des complexes dont les trois groupes R₁ sont identiques et sont des groupes carboxylates.

The photophysical properties of several compounds according to the invention have been compared and the following elements have been discovered:

• the photophysical properties of a symmetrical europium complex (three chromophores chrom ₁ chromium ₂ and chrom ₃ identical) are the same as those of an asymmetric europium complex (two identical chromophores and a chromophore comprising a group-LG allowing the coupling with a molecule to be labeled).
• The photophysical properties of the complexes of europium phenylphosphinates (R ₁ = -PO (OH) -C ₆ H ₅ ), methylphosphinates (R ₁ = -PO (OH) CH ₃ )) and carboxylates (R ₁ = -COO ^- ) are Similar.
• Hybrid europium complexes whose chromophores comprise different groups R ₁ , for example in which one of the three groups R ₁ is a carboxylate group and the two other groups R ₁ are phenylphosphinate groups or methylphosphinates, have photophysical characteristics identical to those complexes whose three groups R ₁ are identical and are carboxylate groups.

Thus, the results obtained in Example 3, namely the fact that these compounds are particularly suitable for laser excitation at 337 nm, can be generalized to the other compounds according to the invention, that is to say to the europium asymmetric and comprising various R ₁ groups (carboxylates or phenylphosphinates derivatives).

Claims (21)

1. A complexing agent of formula (I):

   

   wherein:

   - either a=b=c=1, or a=b=c=0, or a=1 and b=c=0, or a=b=1 and c=0;

   - R₃, R₄ and R₅ are each selected from the following groups or atoms: H, -L-G;

   - Chrom₁, Chrom₂ and Chrom₃ are identical or different chromophores and all three correspond to only one of the following formulae:

   

   

   

   in which

   - d is an integer ranging from 1 to 3;

   - each R₁ is selected from the following groups: -COOH, -PO(OH)R₆, R₆ representing a group selected from: phenyl, benzyl, methyl, ethyl, propyl, n-butyl, sec-butyl, isobutyl, tert-butyl;

   - R₂₁ and R₂₂ represent, independently of each other, a hydrogen atom or a linear or branched alkyl comprising from 1 to 6 carbon atoms;

   - each R₂ is selected from the following groups:

   • linear or branched alkyl groups comprising from 1 to 6 carbon atoms optionally substituted with a PEG group;

   • O-donating groups selected from: OH; -OPhenyl; -O-CH₂-CO-N-Alk; -O-CH₂-CO-O-Alk; -O-CH₂-CO-NH; -O-CH₂-CO-OH; O-CH₂-CO-N-LG; -O-CH₂-CO-O-L-G; -O-L-G; -O-Alk; -O-PEG; the group of formula:

   

   in which z is an integer in the range from 1 to 5;

   • S-donating groups selected from: -S-L-G; -S-Alk; -S-PEG; the group of formula:

   

   in which z is an integer in the range from 1 to 5;

   • NHCO-donating groups selected from: -NHCO-L-G; -NHCO(OAlk); -NHCO(NHAlk); -NHCO(NAlk₁Alk₂); -NHCO(SAlk); -NHCO(Alk); -NHCO-PEG; -NHCO-phenyl; a group of formula:

   

   • SCO-donating groups selected from: -SCO-L-G; -SCO(OAlk); -SCO(NHAlk); -SCO(NAlk₁Alk₂); -SCO(SAlk); -SCO(Alk); -SCO-PEG;

   • NHCS-donating groups selected from: -NHCS(OAlk); -NHCS(NHAlk); -NHCS(NAlk₁Alk₂); -NHCS(SAlk); -NHCS(Alk); -NHCS-PEG; -NHCS-L-G;

   • SCS-donating groups selected from: -SCS(OAlk); -SCS(NHAlk); -SCS(NAlk₁Alk₂); -SCS(SAlk); -SCS(Alk); -SCS-PEG; -SCS-L-G;

   - PEG is a group of formula -CH₂-(CH₂OCH₂)y-CH₂OCH₃, y being an integer ranging from 1 to 5;

   - Alk, Alk₁ and Alk₂ are each a linear or branched C₁-C₁₀ alkyl optionally substituted with an -O-PEG group;

   - L is a spacer arm selected from the following groups:

   1)

   

   2)

   

   3)

   

   4)

   

   5)

   

   6)

   

   7)

   

   8)

   

   9)

   

   10)

   

   11)

   

   12)

   

   13)

   

   in which n, m, p, r are integers from 1 to 16;

   - G is a reactive group for linking the complex or the complexing agent to a molecule to be labeled, said reactive group being selected from: an acrylamide, an activated amine, an activated ester, an aldehyde, an alkyl halide, an anhydride, an aniline, an azide, an aziridine, a carboxylic acid, a diazoalkane, a haloacetamide, a halotriazine, a hydrazine, an imido ester, an isocyanate, an isothiocyanate, a maleimide, a sulfonyl halide, a thiol, a ketone, an amine, an acid halide, a hydroxysuccinimidyl ester, a hydroxysulfosuccinimidyl ester, an azidonitrophenyl, an azidophenyl, a 3-(2-pyridyldithio)propionamide, a glyoxal, a triazine, an acetylenic group, and a group of formula:

   

   

   

   

   

   in which w is in the range from 0 to 8 and v is equal to 0 or 1, and Ar is a saturated or unsaturated 5- or 6-membered heterocycle, comprising 1 to 3 heteroatoms, optionally substituted with a halogen atom;

   it being understood that:

   - when R₂₂ and R₂₁ are alkyl groups, d=1 and when one of the groups R₂₂ or R₂₁ is an alkyl group and the other is a hydrogen atom, d=1 or d=2;

   - when R₂ comprises a group -L-G, R₃ = R₄ = R₅ = H;

   - when R₂ does not comprise a group -L-G, either one of the groups R₃, R₄ and R₅ is a group -L-G or R₃ = R₄ = R₅ = H; and

   - when several groups R₂ are present, at least one is in position 4 of the benzene ring.
2. The complexing agent of claim 1, wherein:

   - Chrom₁ Chrom₂ and Chrom₃ are identical or different chromophores and all three correspond to only one of the following formulae:

   
3. The complexing agent of claim 1, of formula (I):

   

   wherein:

   - either a=b=c=1, or a=b=c=0, or a=1 and b=c=0, or a=b=1 and c=0;

   - R₃, R₄ and R₅ are each selected from the following groups or atoms: H, -L-G;

   - Chrom₁ Chrom₂ and Chrom₃ are identical or different chromophores of formula (II):

   

   wherein

   - d is an integer in the range from 1 to 3;

   - R₁ is chosen from the following groups: -COOH, -PO(OH)R₆, R₆ representing a group selected from: phenyl, benzyl, methyl, ethyl, propyl, n-butyl, sec-butyl, isobutyl, tert-butyl;

   - each R₂ is selected from the following groups:

   • linear or branched alkyl groups comprising from 1 to 6 carbon atoms optionally substituted with a PEG group;

   • O-donating groups selected from: -O-L-G; -O-Alk; -O-PEG; the group of formula:

   

   in which z is an integer in the range from 1 to 5;

   • S-donating groups selected from: -S-L-G; -S-Alk; -S-PEG; the group of formula:

   

   in which z is an integer in the range from 1 to 5;

   • NHCO-donating groups selected from: -NHCO-L-G; -NHCO(OAlk); -NHCO(NHAlk); -NHCO(NAlk₁Alk₂); -NHCO(SAlk); -NHCO(Alk); -NHCO-PEG; the group of formula:

   

   • SCO-donating groups selected from: -SCO-L-G; -SCO(OAlk); -SCO(NHAlk); -SCO(NAlk₁Alk₂); -SCO(SAlk); -SCO(Alk); -SCO-PEG;

   • NHCS-donating groups selected from: -NHCS(OAlk); -NHCS(NHAlk); -NHCS(NAlk₁Alk₂); -NHCS(SAlk); -NHCS(Alk); -NHCS-PEG; -NHCS-L-G;

   • SCS-donating groups selected from: -SCS(OAlk); -SCS(NHAlk); -SCS(NAlk₁Alk₂); -SCS(SAlk); -SCS(Alk); -SCS-PEG; -SCS-L-G;

   - PEG is a group of formula -CH₂-(CH₂OCH₂)y-CH₂OCH₃, y being an integer in the range from 1 to 5;

   - Alk, Alk₁ and Alk₂ are each a linear or branched C₁-C₁₀ alkyl optionally substituted with an -O-PEG group;

   - L is a spacer arm as defined in claim 1;

   - G is a reactive group as defined in claim 1, for linking the complex or the complexing agent to a molecule to be labeled;

   it being understood that:

   - when R₂ comprises a group -L-G, R₃ = R₄ = R₅ = H;

   - when R₂ does not comprise a group -L-G, either one of the groups R₃, R₄ and R₅ is a group -L-G, or R₃ = R₄ = R₅ = H; and

   - when several groups R₂ are present, at least one is in position 4 of the benzene ring.
4. The complexing agent of any one of the preceding claims, characterized in that:

   either two of the groups chrom₁, chrom₂ and chrom₃ are identical and are each substituted with one to three groups R₂ not comprising a group -L-G and the third chromophore is substituted with a group R₂ comprising a group -L-G, the groups R₃-R₅ are hydrogen atoms;

   or chrom₁, chrom₂ and chrom₃ are identical and are each substituted with one to three groups R₂ not comprising a group -L-G, and the groups R₃-R₅ are hydrogen atoms;

   or chrom₁, chrom₂ and chrom₃ are identical and are substituted with one to three groups R₂ not comprising a group -L-G, and one of the groups R₃-R₅ is a group L-G, the others being hydrogen atoms.
5. The complexing agent ofany one of the preceding claims, characterized in that one of the groups R₂ comprises a group -L-G.
6. The complexing agent of any one of the preceding claims, characterized in that one of the groups R₃, R₄ or R₅ comprises a group -L-G.
7. The complexing agent of any one of the preceding claims, characterized in that it does not comprise any group -L-G.
8. The complexing agent of any one of the preceding claims, characterized in that a=b=c=0 (1,4,7-triazacyclononane).
9. The complexing agent of any one of the preceding claims, characterized in that the groups R₁ of the chromophores chrom₁, chrom₂ and chrom₃ are -COOH groups.
10. The complexing agent of any one of the preceding claims, characterized in that the groups R₁ of the chromophores chrom₁, chrom₂ and chrom₃ are groups -PO(OH)R₆.
11. The complexing agent of any one of the preceding claims, characterized in that the group R₁ of the chromophore chrom₁ is a -COOH group and the groups R₁ of the chromophores chrom₂ and chrom₃ are groups -PO(OH)R₆.
12. The complexing agent of any one of the preceding claims, characterized in that the group R₁ of the chromophore chrom₁ is a group -PO(OH)R₆ and the groups R₁ of the chromophores chrom₂ and chrom₃ are -COOH groups.
13. The complexing agent of any one of the preceding claims, characterized in that the groups R₂ are O-donating groups selected from: -O-L-G; -O-Alk; -O-PEG; the group of formula:

    
14. The complexing agent of any one of the preceding claims, characterized in that the groups R₂ are NHCO-donating groups selected from: -NHCO-L-G; -NHCO(OAlk); -NHCO(NHAlk); -NHCO(NAlk₁Alk₂); -NHCO(SAlk); -NHCO(Alk); -NHCO-PEG; the group of formula:

    
15. The complexing agent of any one of the preceding claims, characterized in that it comprises at least one PEG group.
16. The complexing agent of any one of the preceding claims, characterized in that the group -L-G, when it is present, consists of a reactive group G selected from: a carboxylic acid, an amine, a succinimidyl ester, a haloacetamide, a hydrazine, an isothiocyanate, a maleimide group, an aliphatic amine, and of a spacer arm L consisting of an alkylene chain comprising from 1 to 5 carbon atoms.
17. A lanthanide complex comprising a complexing agent of any one of the preceding claims and a lanthanide.
18. The lanthanide complex of claim 19, characterized in that the lanthanide is selected from: Eu³⁺, Tb³⁺, Gd³⁺, Dy³⁺, Nd³⁺, Er³⁺, preferably the lanthanide is Eu³⁺.
19. A fluorescent conjugate of a molecule to be labeled, obtained by placing a lanthanide complex of one of claims 17 and 18, said complex comprising a group -L-G, in contact with a molecule of interest.
20. A method for synthesizing mono-protected trinitrogenous macrocycles, characterized in that it comprises the following steps:

    (i) protection of two of the three amines of the trinitrogenous macrocycle with a hydrogenation-sensitive protecting group Pg₁;

    (ii) purification of the disubstituted compounds on a column selected from: a basic alumina column; a silica column which has undergone a pretreatment with triethylamine or ammonia; a neutral alumina column, the latter being preferred;

    (iii) protection of the third amine of said trinitrogenous macrocycle with a second protecting group Pg₂ which is hydrogenation-insensitive;

    (iv) deprotection of the amines protected with the protecting groups Pg₁ by hydrogenation, preferably with Pearlman's catalyst;

    (v) purification of the monoprotected products, for example by formation of dihydrochloride salts by adding cold hydrochloric acid and precipitation from diethyl ether;

    Pg₁ and Pg₂ being selected from the following groups: Carbobenzyloxy (Cbz), p-Methoxybenzylcarbonyl (Moz) tert-Butyloxycarbonyl (Boc), 9-Fluorenylmethyloxycarbonyl (Fmoc), Acetyl (Ac), Benzoyl (Bz), Benzyl (Bn), p-Methoxybenzyl (PMB), 3,4-Dimethoxybenzyl (DMPM), p-methoxyphenyl (PMP), Tosyl (Ts), Nosyl (Ns), trifluoroacetamides, alkoxycarbonyls, allyloxycarbonyl (Aloc), 2-(trimethylsilyl)ethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl (Troc), 2-(trimethylsilyl)ethoxycarbonyl (Teoc), B-(trimethylsilyl)ethanesulfonyl (SES), benzhydryl, trityl, 9-phenylfluorenyl and N-silyl imines.
21. A dihydrochloride salt of a compound of formula:

    

    wherein

    Pg₂ is a hydrogenation-insensitive protecting group as defined in claim 20;

    a, b and c have the same values as in claim 1.

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